Indolinone compounds and uses thereof

ABSTRACT

Indolinone derivative compounds that act as EWS-FLI1 transcription factor inhibitors are provided. Also provided are pharmaceutical compositions of the indolinone derivatives, methods of synthesizing the same, methods of treating using same, and assays for identifying the inhibitors of EWS-FLI1 oncoprotein.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/062,086, filed Oct. 9, 2014, entitled “INDOLINONE COMPOUNDS AND USESTHEREOF” which is incorporated herein by reference in its entirety.

BACKGROUND

1. Field

Indolinone derivative compounds that act as EWS-FLI1 transcriptionfactor inhibitors are provided. Also provided are pharmaceuticalcompositions of the indolinone derivatives, methods of synthesizing thesame, methods of treating using same, and assays for identifying theinhibitors of EWS-FLI1 oncoprotein.

2. Description

The EWS-FLI transcription factor present in vast variety of Ewing'ssarcoma family of tumors (ESFT) was characterized over ten years ago.Progress in the treatment of Ewing's sarcoma the second most common bonetumor in children and adolescents, has improved survival for patientswith localized tumors. However, patients with metastases still farebadly and the therapy carries short and long-term toxicities. The Ewingsarcoma family of tumors (ESFT) is characterized by a chromosomaltranslocation that generates EWS-FLI1, on oncogenic fusion transcriptionfactor whose continued expression is believed to be critical for ESFTcell survival (Balamuth, N J, Womer, R B., Lancet Oncology 11, 184-192(2010)).

In vitro and in vivo studies have demonstrated that the inhibition ofthe binding of the oncoprotein, EWS-FLI1, to RNA Helicase A (RHA) leadsto a decrease in proliferation of ESFT cell lines and a decrease oftumor volume. EWS-FLI1 lacks enzymatic activity, however, theprotein-protein interaction between RNA helicase A (RHA) andEWS-FLI1-modulates oncogenesis, and is therefore required for themaintenance of the tumor growth (Hyariye N Erkizan et al. NatureMedicine 15(7) 750-756 (2009)). The paradigm of disrupting key proteininteractions may have utility in treatment of diseases includingsarcomas with similar translocations, and leukemias with MLLtranslocations ((Helman L J, Meltzer P. Mechanisms of sarcomadevelopment. Nat Rev Cancer 2003; 3(9):685-94); and Pui C H, et al., NEngl J Med 2004; 350(15):1535-48). Moreover, disordered proteins may beexcellent therapeutic targets based on their intrinsic biochemicalproperties (Cheng Y, LeGall T, Oldfield C J, et al., Trends Biotechnol2006; 24(10):435-42).

SUMMARY

Despite years of in vitro and xenograft studies with antisense and siRNAdirected towards EWS-FLI1, none of these is heretofore practical as ahuman therapy based on inadequate delivery and stability. Accordingly,there is a need for improved therapies to treat disorders such as ESFTs.

FLI-1 is a member of the ETS family transcription factors which arenormally active in the developing embryo, but not after birth. There are29 members of this family of transcription factors, four of which,FLI-1, ETV1, ETV4 and ERG, have been associated with a wide range ofcancers.

Therapeutic compounds targeting the inhibition of the binding ofoncogenic fusion proteins of FLI1, ETV1, ETV4 or ERG or thetranscription factors themselves will have utility in treatment ofcancers including the Ewing's sarcoma family of tumors, pancreaticcancer, prostate cancer, glioblastoma, non-small cell lung cancer, andseveral other cancers. The preferred embodiments fulfill these needs,and provide other advantages as well.

Some embodiments disclosed herein relate to a compound of Formula (I)including forms such as stereoisomers, free forms, pharmaceuticallyacceptable salts or esters thereof, solvates, or combinations of suchforms, wherein A, D, R₁, R₂, R₃, R₄, R₅, R₆, and R₁₂ are as definedherein.

Some embodiments disclosed herein relate to methods for treating cancerin a mammal, comprising administering to the mammal an effective amountof one or more compounds of Formula (I) including forms such asstereoisomers, free forms, or a pharmaceutically acceptable saltthereof, or a pharmaceutical composition that includes one or morecompounds of Formula (I) including forms such as stereoisomers, freeforms, or a pharmaceutically acceptable salt thereof. Other embodimentsdescribed herein relate to using one or more compounds of Formula (I)including forms such as stereoisomers, free forms, or a pharmaceuticallyacceptable salt thereof, in the manufacture of a medicament fortreatment of cancer.

Still other embodiments described herein relate to a compound of Formula(I) including forms such as stereoisomers, free forms, or apharmaceutically acceptable salt thereof, for treatment of cancerwherein the cancer is selected from the group consisting of Ewing'ssarcoma, glioblastoma, acute myeloid leukemia, breast cancer, head &neck cancer, melanoma, non-small cell lung cancer, ovarian cancer,prostate cancer, and uterine cancer. These and other embodiments aredescribed in greater detail below.

DETAILED DESCRIPTION

The following description and examples illustrate a preferred embodimentof the present invention in detail. Those of skill in the art willrecognize that there are numerous variations and modifications of thisinvention that are encompassed by its scope. Accordingly, thedescription of a preferred embodiment should not be deemed to limit thescope of the present invention

Chromosomal translocations generating oncogenic transcription factorsare the hallmark of a variety of tumors, including many sarcomas. Ewingsarcoma family of tumors (ESFTs) are characterized by thet(11;22)(q24;q12) translocation that generates the Ewing sarcomabreakpoint region 1 and Friend leukemia virus integration 1 (EWS-FLI1)fusion transcription factor responsible for the highly malignantphenotype of this tumor. Continued expression of EWS-FLI1 is believed tobe critical for ESFT cell survival. EWS-FLI1 is an attractive treatmenttarget for Ewing sarcoma because of its malignant cell specificity.Furthermore, experimental evidence indicates that EWS/FLI expression isessential for Ewing sarcoma tumor cells. In vitro targeting of EWS-FLI1with antisense oligodeoxynucleotides and RNA interference (RNAi)inhibits Ewing sarcoma cell viability, growth, and oncogenictransformation, supporting EWS-FLI1 attenuation as a potential treatmentmodality. The therapeutic agents of the preferred embodiments have broadapplicability to a larger group of tumors, and are useful astherapeutics for treatment for other oncogenic transcription factorrelated malignancies such as chemotherapy-resistant sarcomas andleukemias and difficult to treat tumors such as Ewing's sarcoma.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art. All patents, applications, published applications and otherpublications referenced herein are incorporated by reference in theirentirety unless stated otherwise. In the event that there is a pluralityof definitions for a term herein, those in this section prevail unlessstated otherwise.

As used herein, any “R” group(s) such as, without limitation, R₁, R₂,R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁, and R₁₂ represent substituentsthat can be attached to the indicated atom. An R group may besubstituted or unsubstituted. If two “R” groups are described as being“taken together” the R groups and the atoms they are attached to canform a cycloalkyl, cycloalkenyl, aryl, heteroaryl or heterocycle. Forexample, without limitation, if R^(a) and R^(b) of an NR^(a)R^(b) groupare indicated to be “taken together,” it means that they are covalentlybonded to one another to form a ring:

In addition, if two “R” groups are described as being “taken together”with the atom(s) to which they are attached to form a ring as analternative, the R groups may not be limited to the variables orsubstituents defined previously.

As used herein, “alkyl” refers to a straight or branched hydrocarbonchain that comprises a fully saturated (no double or triple bonds)hydrocarbon group. The alkyl group may have 1 to 20 carbon atoms(whenever it appears herein, a numerical range such as “1 to 20” refersto each integer in the given range; e.g., “1 to 20 carbon atoms” meansthat the alkyl group may consist of 1 carbon atom, 2 carbon atoms, 3carbon atoms, etc., up to and including 20 carbon atoms, although thepresent definition also covers the occurrence of the term “alkyl” whereno numerical range is designated). The alkyl group may also be a mediumsize alkyl having 1 to 10 carbon atoms. The alkyl group could also be alower alkyl having 1 to 6 carbon atoms. The alkyl group of the compoundsmay be designated as “C₁-C₆ alkyl” or similar designations. By way ofexample only, “C₁-C₆ alkyl” indicates that there are one to six carbonatoms in the alkyl chain, i.e., the alkyl chain is selected from methyl,ethyl, propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, and t-butyl,pentyl (straight and branched) and hexyl (straight and branched).Typical alkyl groups include, but are in no way limited to, methyl,ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl(straight and branched) and hexyl (straight and branched). The alkylgroup may be substituted or unsubstituted.

As used herein, “cycloalkyl” refers to a completely saturated (no doubleor triple bonds) mono- or multi-cyclic hydrocarbon ring system. Whencomposed of two or more rings, the rings may be joined together in afused fashion. Cycloalkyl groups can contain 3 to 10 atoms in thering(s) or 3 to 8 atoms in the ring(s). A cycloalkyl group may beunsubstituted or substituted. Typical cycloalkyl groups include, but arein no way limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl and cyclooctyl.

As used herein, “aryl” refers to a carbocyclic (all carbon) mono-cyclicor multi-cyclic aromatic ring system (including fused ring systems wheretwo carbocyclic rings share a chemical bond) that has a fullydelocalized pi-electron system throughout all the rings. The number ofcarbon atoms in an aryl group can vary. For example, the aryl group canbe a C₆-C₁₄ aryl group, a C₆-C₁₀ aryl group, or a C₆ aryl group.Examples of aryl groups include, but are not limited to, benzene,naphthalene and azulene. An aryl group may be substituted orunsubstituted.

As used herein, “heteroaryl” refers to a mono-cyclic or multi-cyclicaromatic ring system (a ring system with fully delocalized pi-electronsystem) that contain(s) one or more heteroatoms (for example, 1 to 5heteroatoms), that is, an element other than carbon, including but notlimited to, nitrogen, oxygen and sulfur. The number of atoms in thering(s) of a heteroaryl group can vary. For example, the heteroarylgroup can contain 4 to 14 atoms in the ring(s), 5 to 10 atoms in thering(s) or 5 to 6 atoms in the ring(s). Furthermore, the term“heteroaryl” includes fused ring systems where two rings, such as atleast one aryl ring and at least one heteroaryl ring, or at least twoheteroaryl rings, share at least one chemical bond. Examples ofheteroaryl rings include, but are not limited to, furan, furazan,thiophene, benzothiophene, phthalazine, pyrrole, oxazole, benzoxazole,1,2,3-oxadiazole, 1,2,4-oxadiazole, thiazole, 1,2,3-thiadiazole,1,2,4-thiadiazole, benzothiazole, imidazole, benzimidazole, indole,indazole, pyrazole, benzopyrazole, isoxazole, benzoisoxazole,isothiazole, triazole, benzotriazole, thiadiazole, tetrazole, pyridine,pyridazine, pyrimidine, pyrazine, purine, pteridine, quinoline,isoquinoline, quinazoline, quinoxaline, cinnoline and triazine. Aheteroaryl group may be substituted or unsubstituted.

As used herein, heterocycloalkyl refers to three-, four-, five-, six-,seven-, eight-, nine-, ten-, up to 18-membered mono-cyclic, bicyclic,and tricyclic ring system wherein carbon atoms together with from 1 to 5heteroatoms constitute said ring system. A heterocycle may optionallycontain one or more unsaturated bonds situated in such a way, however,that a fully delocalized pi-electron system does not occur throughoutall the rings. The heteroatom(s) is an element other than carbonincluding, but not limited to, oxygen, sulfur, and nitrogen. Aheterocycle may further contain one or more carbonyl or thiocarbonylfunctionalities, so as to make the definition include oxo-systems andthio-systems such as lactams, lactones, cyclic imides, cyclic thioimidesand cyclic carbamates. When composed of two or more rings, the rings maybe joined together in a fused fashion. Additionally, any nitrogens in aheterocycloalky may be quaternized. Heterocycloalkyl groups may beunsubstituted or substituted. Examples of such heterocycloalkyl groupsinclude but are not limited to, 1,3-dioxin, 1,3-dioxane, 1,4-dioxane,1,2-dioxolane, 1,3-dioxolane, 1,4-dioxolane, 1,3-oxathiane,1,4-oxathiin, 1,3-oxathiolane, 1,3-dithiole, 1,3-dithiolane,1,4-oxathiane, tetrahydro-1,4-thiazine, 2H-1,2-oxazine, maleimide,succinimide, barbituric acid, thiobarbituric acid, dioxopiperazine,hydantoin, dihydrouracil, trioxane, hexahydro-1,3,5-triazine,imidazoline, imidazolidine, isoxazoline, isoxazolidine, oxazoline,oxazolidine, oxazolidinone, thiazoline, thiazolidine, morpholine,oxirane, piperidine N-Oxide, piperidine, piperazine, pyrrolidine,pyrrolidone, pyrrolidione, 4-piperidone, pyrazoline, pyrazolidine,2-oxopyrrolidine, tetrahydropyran, 4H-pyran, tetrahydrothiopyran,thiamorpholine, thiamorpholine sulfoxide, thiamorpholine sulfone, andtheir benzo-fused analogs (e.g., benzimidazolidinone,tetrahydroquinoline and 3,4-methylenedioxyphenyl).

The term “pharmaceutically acceptable salt” refers to a salt of acompound that does not cause significant irritation to an organism towhich it is administered and does not abrogate the biological activityand properties of the compound. In some embodiments, the salt is an acidaddition salt of the compound. Pharmaceutical salts can be obtained byreacting a compound with inorganic acids such as hydrohalic acid (e.g.,hydrochloric acid or hydrobromic acid), sulfuric acid, nitric acid andphosphoric acid. Pharmaceutical salts can also be obtained by reacting acompound with an organic acid such as aliphatic or aromatic carboxylicor sulfonic acids, for example formic, acetic, succinic, lactic, malic,tartaric, citric, ascorbic, nicotinic, methanesulfonic, ethanesulfonic,p-toluensulfonic, salicylic or naphthalenesulfonic acid. Pharmaceuticalsalts can also be obtained by reacting a compound with a base to form asalt such as an ammonium salt, an alkali metal salt, such as a sodium ora potassium salt, an alkaline earth metal salt, such as a calcium or amagnesium salt, a salt of organic bases such as dicyclohexylamine,N-methyl-D-glucamine, tris(hydroxymethyl)methylamine, C₁-C₇ alkylamine,cyclohexylamine, triethanolamine, ethylenediamine, and salts with aminoacids such as arginine and lysine.

It is understood that, in any compound described herein having one ormore chiral centers, if an absolute stereochemistry is not expresslyindicated, then each center may independently be of R-configuration orS-configuration or a mixture thereof. Thus, the compounds providedherein may be enantiomerically pure, enantiomerically enriched, racemicmixture, diastereomerically pure, diastereomerically enriched, or astereoisomeric mixture. In addition it is understood that, in anycompound described herein having one or more double bond(s) generatinggeometrical isomers that can be defined as E or Z, each double bond mayindependently be E or Z a mixture thereof.

It is to be understood that where compounds disclosed herein haveunfilled valencies, then the valencies are to be filled with hydrogensor isotopes thereof, e.g., hydrogen-1 (protium) and hydrogen-2(deuterium).

It is understood that the compounds described herein can be labeledisotopically. Substitution with isotopes such as deuterium may affordcertain therapeutic advantages resulting from greater metabolicstability, such as, for example, increased in vivo half-life or reduceddosage requirements. Each chemical element as represented in a compoundstructure may include any isotope of said element. For example, in acompound structure a hydrogen atom may be explicitly disclosed orunderstood to be present in the compound. At any position of thecompound that a hydrogen atom may be present, the hydrogen atom can beany isotope of hydrogen, including but not limited to hydrogen-1(protium) and hydrogen-2 (deuterium). Thus, reference herein to acompound encompasses all potential isotopic forms unless the contextclearly dictates otherwise.

It is understood that the methods and combinations described hereininclude crystalline forms (also known as polymorphs, which include thedifferent crystal packing arrangements of the same elemental compositionof a compound), amorphous phases, salts, solvates, and hydrates. In someembodiments, the compounds described herein exist in solvated forms withpharmaceutically acceptable solvents such as water, ethanol, or thelike. In other embodiments, the compounds described herein exist inunsolvated form. Solvates contain either stoichiometric ornon-stoichiometric amounts of a solvent, and may be formed during theprocess of crystallization with pharmaceutically acceptable solventssuch as water, ethanol, or the like. Hydrates are formed when thesolvent is water, or alcoholates are formed when the solvent is alcohol.In addition, the compounds provided herein can exist in unsolvated aswell as solvated forms. In general, the solvated forms are consideredequivalent to the unsolvated forms for the purposes of the compounds andmethods provided herein.

Where a range of values is provided, it is understood that the upper andlower limit, and each intervening value between the upper and lowerlimit of the range is encompassed within the embodiments.

Compounds

In a first aspect a compound is provided having Formula (I):

or a stereoisomer, a pharmaceutically acceptable salt, or solvatethereof, wherein R₁, R₂, R₃, and R₄ are independently selected from thegroup consisting of H, Cl, —CN and —CF₃; wherein A is selected from thegroup consisting of H and C₁₋₆ alkyl; wherein D is selected from thegroup consisting of —OH and —O(C₁₋₆ alkyl); wherein R₅ and R₆ areindependently selected from the group consisting of H, F, and C₁₋₆alkyl, or wherein R₅ and R₆ taken together form a substituted orunsubstituted cycloalkyl ring; wherein R₁₂ is independently selectedfrom the group consisting of C₃₋₈ cycloalkyl and

wherein R₇, R₈, R₉, R₁₀ and R₁₁ are independently selected from thegroup consisting of H, halogen, CN, CF₃, C₁₋₆ alkyl, aryl, heteroaryl,—O(aryl), —O(heteroaryl), —CO₂H, —CO₂(C₁₋₆ alkyl), —NHSO₂(C₁₋₆ alkyl),—NHSO₂(aryl), —NHCONH(C₁₋₆ alkyl), —NHCON(C₁₋₆ alkyl)₂, —N(C₁₋₆alkyl)CONH₂, —N(C₁₋₆ alkyl)CONH(C₁₋₆ alkyl), —N(C₁₋₆ alkyl)CON(C₁₋₆alkyl)₂, —SO₂(C₁₋₆ alkyl), —SO₂NH₂, —SO₂NH(C₁₋₆ alkyl), —SO₂N(C₁₋₆alkyl)₂, C₃₋₈ cycloalkyl, and C₃₋₈ heterocycloalkyl.

In an embodiment of the first aspect, R₁, R₂, R₃, and R₄ areindependently selected from the group consisting of hydrogen and Cl.

In an embodiment of the first aspect, R₅ and R₆ taken together form asubstituted or unsubstituted cycloalkyl ring.

In an embodiment of the first aspect, A is H.

In an embodiment of the first aspect, D is OH.

In an embodiment of the first aspect, A is H and D is OH.

In an embodiment of the first aspect, R₉ is selected from the groupconsisting of aziridinyl, azetidinyl, pyrrolidinyl, and morpholinyl.

In an embodiment of the first aspect, R₉ is selected from the groupconsisting of isopropyl and cyclopropyl.

In an embodiment of the first aspect, a compound having a structure ofFormula (Ia):

or a stereoisomer, a pharmaceutically acceptable salt, or solvatethereof, wherein R₁, R₂, R₃, and R₄ are independently selected from thegroup consisting of H and Cl; wherein R₇, R₈, R₁₀ and R₁₁ areindependently selected from the group consisting of H and halogen; andwherein R₉ is independently selected from the group consisting C₃₋₈cycloalkyl and C₃₋₈ heterocycloalkyl.

In an embodiment of the first aspect, R₁ and R₄ are Cl and R₂ and R₃ areH.

In an embodiment of the first aspect, the compound of Formula (I) isselected from the group consisting of:

or a stereoisomer, or a pharmaceutically acceptable salt, ester, orsolvate thereof.

In an embodiment of the first aspect, the compound selected from thegroup consisting of:

or a stereoisomer, a pharmaceutically acceptable salt, ester, or solvatethereof.

In a second aspect, a pharmaceutical composition comprising the compoundof any embodiment of the first aspect or any embodiment thereof and apharmaceutically acceptable carrier is provided.

In a third aspect, a pharmaceutical composition comprising the compoundof any embodiment of the first aspect or any embodiment thereof and apharmaceutically acceptable excipient is provided.

In a fourth aspect, a pharmaceutical composition comprising the compoundof any embodiment of the first aspect or any embodiment thereof and atleast one additional pharmaceutically active agent.

In a fifth aspect, a method for treating cancer is provided comprisingadministering an effective amount of the compound of the first aspect orany embodiment thereof to a subject in need thereof.

In an embodiment of the fifth aspect, the subject is mammalian.

In an embodiment of the fifth aspect, the subject is human.

In an embodiment of the fifth aspect, the cancer is selected from thegroup consisting of Ewing's sarcoma, prostate cancer, glioblastoma,acute myeloid leukemia, breast cancer, head & neck cancer, melanoma,non-small cell lung cancer, ovarian cancer, and uterine cancer.

In a sixth aspect, a method of killing or inhibiting the growth of aneoplastic cell is provided, comprising contacting the cell with aneffective amount of the compound of the first aspect or any embodimentthereof.

In an embodiment of the sixth aspect, the cell is mammalian.

In an embodiment of the sixth aspect, the cell is human.

In an embodiment of the sixth aspect, the cell is in vitro.

In an embodiment of the sixth aspect, the cell is in vivo.

In an embodiment of the sixth aspect, the cell is a cancer cell, thecancer being selected from the group consisting of Ewing's sarcoma,prostate cancer, glioblastoma, acute myeloid leukemia, breast cancer,head & neck cancer, melanoma, non-small cell lung cancer, ovariancancer, and uterine cancer.

In a seventh aspect, a method for inhibiting proliferation of a cell,wherein the cell overexpresses an ETS gene or comprises an ETS fusiongene, comprising contacting the cell with an effective amount of thecompound of the first aspect or any embodiment thereof.

In an embodiment of the seventh aspect, the ETS gene or the ETS fusiongene is selected from the group consisting of FLI1, ERG, ETV1, and ETV4.

In an embodiment of the seventh aspect, the cell is mammalian.

In an embodiment of the seventh aspect, the cell is human.

In an embodiment of the seventh aspect, the cell is in vitro.

In an embodiment of the seventh aspect, the cell is in vivo.

In an embodiment of the seventh aspect, the cell is a cancer cell, thecancer being selected from the group consisting of Ewing's sarcoma,prostate cancer, glioblastoma, acute myeloid leukemia, breast cancer,head & neck cancer, melanoma, non-small cell lung cancer, ovariancancer, and uterine cancer.

Synthetic Methods

Compounds of Formula (I) described herein may be prepared in variousways. General synthetic routes to compounds of Formula (I) are shown anddescribed herein. The routes shown and described herein are illustrativeonly and are not intended, nor are they to be construed, to limit thescope of the claims in any manner whatsoever. Those skilled in the artwill be able to recognize modifications of the disclosed syntheses andto devise alternate routes based on the disclosures herein; all suchmodifications and alternate routes are within the scope of the claims.

Depending upon the substituents present, the small molecule inhibitorscan be in a form of a pharmaceutically acceptable salt. The terms“pharmaceutically acceptable salt” as used herein are broad terms, andis to be given its ordinary and customary meaning to a person ofordinary skill in the art (and is not to be limited to a special orcustomized meaning), and refers without limitation to salts preparedfrom pharmaceutically acceptable, non-toxic acids or bases. Suitablepharmaceutically acceptable salts include metallic salts, e.g., salts ofaluminum, zinc, alkali metal salts such as lithium, sodium, andpotassium salts, alkaline earth metal salts such as calcium andmagnesium salts; organic salts, e.g., salts of lysine,N,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,ethylenediamine, meglumine (N-methylglucamine), procaine, and tris;salts of free acids and bases; inorganic salts, e.g., sulfate,hydrochloride, and hydrobromide; and other salts which are currently inwidespread pharmaceutical use and are listed in sources well known tothose of skill in the art, such as, for example, The Merck Index. Anysuitable constituent can be selected to make a salt of the therapeuticagents discussed herein, provided that it is non-toxic and does notsubstantially interfere with the desired activity.

The compounds of preferred embodiments can include isomers, racemates,optical isomers, enantiomers, diastereomers, tautomers, and cis/transconformers. All such isomeric forms are included within preferredembodiments, including mixtures thereof. As discussed above, thecompounds of preferred embodiments may have chiral centers, for example,they may contain asymmetric carbon atoms and may thus exist in the formof enantiomers or diastereoisomers and mixtures thereof, e.g.,racemates. Asymmetric carbon atom(s) can be present in the (R)- or(S)-configuration, or can be present as mixtures of the (R)- and(S)-forms. The following are isomeric forms of the compounds of Formula(I):

The compounds can be in amorphous form, or in crystalline forms. Thecrystalline forms of the compounds of preferred embodiments can exist aspolymorphs, which are included in preferred embodiments. In addition,some of the compounds of preferred embodiments may also form solvateswith water or other organic solvents. Such solvates are similarlyincluded within the scope of the preferred embodiments.

Certain Pharmaceutical Compositions

It is generally preferred to administer the inhibitors of preferredembodiments in an intravenous or subcutaneous unit dosage form; however,other routes of administration are also contemplated. Contemplatedroutes of administration include but are not limited to oral,parenteral, intravenous, and subcutaneous. The inhibitors of preferredembodiments can be formulated into liquid preparations for, e.g., oraladministration. Suitable forms include suspensions, syrups, elixirs, andthe like. Particularly preferred unit dosage forms for oraladministration include tablets and capsules. Unit dosage formsconfigured for administration once a day are particularly preferred;however, in certain embodiments it can be desirable to configure theunit dosage form for administration twice a day, or more.

The pharmaceutical compositions of preferred embodiments are preferablyisotonic with the blood or other body fluid of the recipient. Theisotonicity of the compositions can be attained using sodium tartrate,propylene glycol or other inorganic or organic solutes. Sodium chlorideis particularly preferred. Buffering agents can be employed, such asacetic acid and salts, citric acid and salts, boric acid and salts, andphosphoric acid and salts. Parenteral vehicles include sodium chloridesolution, Ringer's dextrose, dextrose and sodium chloride, lactatedRinger's or fixed oils. Intravenous vehicles include fluid and nutrientreplenishers, electrolyte replenishers (such as those based on Ringer'sdextrose), and the like.

Viscosity of the pharmaceutical compositions can be maintained at theselected level using a pharmaceutically acceptable thickening agent.Methylcellulose is preferred because it is readily and economicallyavailable and is easy to work with. Other suitable thickening agentsinclude, for example, xanthan gum, carboxymethyl cellulose,hydroxypropyl cellulose, carbomer, and the like. The preferredconcentration of the thickener will depend upon the thickening agentselected. An amount is preferably used that will achieve the selectedviscosity. Viscous compositions are normally prepared from solutions bythe addition of such thickening agents.

A pharmaceutically acceptable preservative can be employed to increasethe shelf life of the pharmaceutical compositions. Benzyl alcohol can besuitable, although a variety of preservatives including, for example,parabens, thimerosal, chlorobutanol, or benzalkonium chloride can alsobe employed. A suitable concentration of the preservative is typicallyfrom about 0.02% to about 2% based on the total weight of thecomposition, although larger or smaller amounts can be desirabledepending upon the agent selected. Reducing agents, as described above,can be advantageously used to maintain good shelf life of theformulation.

The inhibitors of preferred embodiments can be in admixture with asuitable carrier, diluent, or excipient such as sterile water,physiological saline, glucose, or the like, and can contain auxiliarysubstances such as wetting or emulsifying agents, pH buffering agents,gelling or viscosity enhancing additives, preservatives, flavoringagents, colors, and the like, depending upon the route of administrationand the preparation desired. See, e.g., “Remington: The Science andPractice of Pharmacy”, Lippincott Williams & Wilkins; 20th edition (Jun.1, 2003) and “Remington's Pharmaceutical Sciences,” Mack Pub. Co.;18^(th) and 19^(th) editions (December 1985, and June 1990,respectively). Such preparations can include complexing agents, metalions, polymeric compounds such as polyacetic acid, polyglycolic acid,hydrogels, dextran, and the like, liposomes, microemulsions, micelles,unilamellar or multilamellar vesicles, erythrocyte ghosts orspheroblasts. Suitable lipids for liposomal formulation include, withoutlimitation, monoglycerides, diglycerides, sulfatides, lysolecithin,phospholipids, saponin, bile acids, and the like. The presence of suchadditional components can influence the physical state, solubility,stability, rate of in vivo release, and rate of in vivo clearance, andare thus chosen according to the intended application, such that thecharacteristics of the carrier are tailored to the selected route ofadministration.

For oral administration, the pharmaceutical compositions can be providedas a tablet, aqueous or oil suspension, dispersible powder or granule,emulsion, hard or soft capsule, syrup or elixir. Compositions intendedfor oral use can be prepared according to any method known in the artfor the manufacture of pharmaceutical compositions and can include oneor more of the following agents: sweeteners, flavoring agents, coloringagents and preservatives. Aqueous suspensions can contain the activeingredient in admixture with excipients suitable for the manufacture ofaqueous suspensions.

Formulations for oral use can also be provided as hard gelatin capsules,wherein the active ingredient(s) are mixed with an inert solid diluent,such as calcium carbonate, calcium phosphate, or kaolin, or as softgelatin capsules. In soft capsules, the inhibitors can be dissolved orsuspended in suitable liquids, such as water or an oil medium, such aspeanut oil, olive oil, fatty oils, liquid paraffin, or liquidpolyethylene glycols. Stabilizers and microspheres formulated for oraladministration can also be used. Capsules can include push-fit capsulesmade of gelatin, as well as soft, sealed capsules made of gelatin and aplasticizer, such as glycerol or sorbitol. The push-fit capsules cancontain the active ingredient in admixture with fillers such as lactose,binders such as starches, and/or lubricants such as talc or magnesiumstearate and, optionally, stabilizers.

Tablets can be uncoated or coated by known methods to delaydisintegration and absorption in the gastrointestinal tract and therebyprovide a sustained action over a longer period of time. For example, atime delay material such as glyceryl monostearate can be used. Whenadministered in solid form, such as tablet form, the solid formtypically comprises from about 0.001 wt. % or less to about 50 wt. % ormore of active ingredient(s), preferably from about 0.005, 0.01, 0.02,0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,0.7, 0.8, 0.9, or 1 wt. % to about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20,25, 30, 35, 40, or 45 wt. %.

Tablets can contain the active ingredients in admixture with non-toxicpharmaceutically acceptable excipients including inert materials. Forexample, a tablet can be prepared by compression or molding, optionally,with one or more additional ingredients. Compressed tablets can beprepared by compressing in a suitable machine the active ingredients ina free-flowing form such as powder or granules, optionally mixed with abinder, lubricant, inert diluent, surface active or dispersing agent.Molded tablets can be made by molding, in a suitable machine, a mixtureof the powdered inhibitor moistened with an inert liquid diluent.

Preferably, each tablet or capsule contains from about 1 mg or less toabout 1,000 mg or more of an inhibitor of the preferred embodiments,more preferably from about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 mgto about 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700,750, 800, or 900 mg. Most preferably, tablets or capsules are providedin a range of dosages to permit divided dosages to be administered. Adosage appropriate to the patient and the number of doses to beadministered daily can thus be conveniently selected. In certainembodiments it can be preferred to incorporate two or more of thetherapeutic agents to be administered into a single tablet or otherdosage form (e.g., in a combination therapy); however, in otherembodiments it can be preferred to provide the therapeutic agents inseparate dosage forms.

Suitable inert materials include diluents, such as carbohydrates,mannitol, lactose, anhydrous lactose, cellulose, sucrose, modifieddextrans, starch, and the like, or inorganic salts such as calciumtriphosphate, calcium phosphate, sodium phosphate, calcium carbonate,sodium carbonate, magnesium carbonate, and sodium chloride.Disintegrants or granulating agents can be included in the formulation,for example, starches such as corn starch, alginic acid, sodium starchglycolate, Amberlite, sodium carboxymethylcellulose, ultramylopectin,sodium alginate, gelatin, orange peel, acid carboxymethyl cellulose,natural sponge and bentonite, insoluble cationic exchange resins,powdered gums such as agar, karaya or tragacanth, or alginic acid orsalts thereof.

Binders can be used to form a hard tablet. Binders include materialsfrom natural products such as acacia, tragacanth, starch and gelatin,methyl cellulose, ethyl cellulose, carboxymethyl cellulose, polyvinylpyrrolidone, hydroxypropylmethyl cellulose, and the like.

Lubricants, such as stearic acid or magnesium or calcium salts thereof,polytetrafluoroethylene, liquid paraffin, vegetable oils and waxes,sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycol,starch, talc, pyrogenic silica, hydrated silicoaluminate, and the like,can be included in tablet formulations.

Surfactants can also be employed, for example, anionic detergents suchas sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctylsodium sulfonate, cationic such as benzalkonium chloride or benzethoniumchloride, or nonionic detergents such as polyoxyethylene hydrogenatedcastor oil, glycerol monostearate, polysorbates, sucrose fatty acidester, methyl cellulose, or carboxymethyl cellulose.

Controlled release formulations can be employed wherein the amifostineor analog(s) thereof is incorporated into an inert matrix that permitsrelease by either diffusion or leaching mechanisms. Slowly degeneratingmatrices can also be incorporated into the formulation. Other deliverysystems can include timed release, delayed release, or sustained releasedelivery systems.

Coatings can be used, for example, nonenteric materials such as methylcellulose, ethyl cellulose, hydroxyethyl cellulose, methylhydroxy-ethylcellulose, hydroxypropyl cellulose, hydroxypropyl-methyl cellulose,sodium carboxy-methyl cellulose, providone and the polyethylene glycols,or enteric materials such as phthalic acid esters. Dyestuffs or pigmentscan be added for identification or to characterize differentcombinations of inhibitor doses

When administered orally in liquid form, a liquid carrier such as water,petroleum, oils of animal or plant origin such as peanut oil, mineraloil, soybean oil, or sesame oil, or synthetic oils can be added to theactive ingredient(s). Physiological saline solution, dextrose, or othersaccharide solution, or glycols such as ethylene glycol, propyleneglycol, or polyethylene glycol are also suitable liquid carriers. Thepharmaceutical compositions can also be in the form of oil-in-wateremulsions. The oily phase can be a vegetable oil, such as olive orarachis oil, a mineral oil such as liquid paraffin, or a mixturethereof. Suitable emulsifying agents include naturally-occurring gumssuch as gum acacia and gum tragacanth, naturally occurring phosphatides,such as soybean lecithin, esters or partial esters derived from fattyacids and hexitol anhydrides, such as sorbitan mono-oleate, andcondensation products of these partial esters with ethylene oxide, suchas polyoxyethylene sorbitan mono-oleate. The emulsions can also containsweetening and flavoring agents.

Pulmonary delivery can also be employed. The compound is delivered tothe lungs while inhaling and traverses across the lung epithelial liningto the blood stream. A wide range of mechanical devices designed forpulmonary delivery of therapeutic products can be employed, includingbut not limited to nebulizers, metered dose inhalers, and powderinhalers, all of which are familiar to those skilled in the art. Thesedevices employ formulations suitable for the dispensing of compound.Typically, each formulation is specific to the type of device employedand can involve the use of an appropriate propellant material, inaddition to diluents, adjuvants, and/or carriers useful in therapy.

The compound and/or other optional active ingredients are advantageouslyprepared for pulmonary delivery in particulate form with an averageparticle size of from 0.1 μm or less to 10 μm or more, more preferablyfrom about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or 0.9 μm to about 1.0,1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0,8.5, 9.0, or 9.5 μm. Pharmaceutically acceptable carriers for pulmonarydelivery of inhibitor include carbohydrates such as trehalose, mannitol,xylitol, sucrose, lactose, and sorbitol. Other ingredients for use informulations can include DPPC, DOPE, DSPC, and DOPC. Natural orsynthetic surfactants can be used, including polyethylene glycol anddextrans, such as cyclodextran. Bile salts and other related enhancers,as well as cellulose and cellulose derivatives, and amino acids can alsobe used. Liposomes, microcapsules, microspheres, inclusion complexes,and other types of carriers can also be employed.

Pharmaceutical formulations suitable for use with a nebulizer, eitherjet or ultrasonic, typically comprise the inhibitor dissolved orsuspended in water at a concentration of about 0.01 or less to 100 mg ormore of inhibitor per mL of solution, preferably from about 0.1, 1, 2,3, 4, 5, 6, 7, 8, 9, or 10 mg to about 15, 20, 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, or 90 mg per mL of solution. The formulationcan also include a buffer and a simple sugar (e.g., for proteinstabilization and regulation of osmotic pressure). The nebulizerformulation can also contain a surfactant, to reduce or prevent surfaceinduced aggregation of the inhibitor caused by atomization of thesolution in forming the aerosol.

Formulations for use with a metered-dose inhaler device generallycomprise a finely divided powder containing the active ingredientssuspended in a propellant with the aid of a surfactant. The propellantcan include conventional propellants, such as chlorofluorocarbons,hydrochlorofluorocarbons, hydrofluorocarbons, and hydrocarbons.Preferred propellants include trichlorofluoromethane,dichlorodifluoromethane, dichlorotetrafluoroethanol,1,1,1,2-tetrafluoroethane, and combinations thereof. Suitablesurfactants include sorbitan trioleate, soya lecithin, and oleic acid.

Formulations for dispensing from a powder inhaler device typicallycomprise a finely divided dry powder containing inhibitor, optionallyincluding a bulking agent, such as lactose, sorbitol, sucrose, mannitol,trehalose, or xylitol in an amount that facilitates dispersal of thepowder from the device, typically from about 1 wt. % or less to 99 wt. %or more of the formulation, preferably from about 5, 10, 15, 20, 25, 30,35, 40, 45, or 50 wt. % to about 55, 60, 65, 70, 75, 80, 85, or 90 wt. %of the formulation.

When a compound of the preferred embodiments is administered byintravenous, parenteral, or other injection, it is preferably in theform of a pyrogen-free, parenterally acceptable aqueous solution oroleaginous suspension. Suspensions can be formulated according tomethods well known in the art using suitable dispersing or wettingagents and suspending agents. The preparation of acceptable aqueoussolutions with suitable pH, isotonicity, stability, and the like, iswithin the skill in the art. A preferred pharmaceutical composition forinjection preferably contains an isotonic vehicle such as1,3-butanediol, water, isotonic sodium chloride solution, Ringer'ssolution, dextrose solution, dextrose and sodium chloride solution,lactated Ringer's solution, or other vehicles as are known in the art.In addition, sterile fixed oils can be employed conventionally as asolvent or suspending medium. For this purpose, any bland fixed oil canbe employed including synthetic mono or diglycerides. In addition, fattyacids such as oleic acid can likewise be used in the formation ofinjectable preparations. The pharmaceutical compositions can alsocontain stabilizers, preservatives, buffers, antioxidants, or otheradditives known to those of skill in the art.

The duration of the injection can be adjusted depending upon variousfactors, and can comprise a single injection administered over thecourse of a few seconds or less, to 0.5, 0.1, 0.25, 0.5, 0.75, 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, or 24 hours or more of continuous intravenous administration.

The compounds of the preferred embodiments can additionally employadjunct components conventionally found in pharmaceutical compositionsin their art-established fashion and at their art-established levels.Thus, for example, the compositions can contain additional compatiblepharmaceutically active materials for combination therapy (such assupplementary antimicrobials, antipruritics, astringents, localanesthetics, anti-inflammatory agents, reducing agents,chemotherapeutics and the like), or can contain materials useful inphysically formulating various dosage forms of the preferredembodiments, such as excipients, dyes, thickening agents, stabilizers,preservatives or antioxidants. Anti-cancer agents that can be used incombination with the compounds of preferred embodiments include, but arenot limited to, vinca alkaloids such as vinblastine and vincristine;anthracyclines such as doxorubicin, daunorubicin, epirubicin;anthracenes such as bisantrene and mitoxantrone; epipodophyllo-toxinssuch as etoposide and teniposide; and other anticancer drugs such asactinomyocin D, mithomycin C, mitramycin, methotrexate, docetaxel,etoposide (VP-16), paclitaxel, docetaxel, and adriamycin); andimmunosuppressants (e.g., cyclosporine A, tacrolimus). In someembodiments, the compounds, compositions and methods provided herein maybe in combination with histone deacetylase inhibitors (HDAC), aurorakinase inhibitors, demethylating agents (such as 5-AZA cytidine),immunotherapy with natural killer cells, IGF-IR antibodies, Ewingantigen antibodies, immunosuppressive drugs, and hydroxyurea. Examplesof histone deacetylase inhibitors include vorinostat, romidepsin,panobinostat, valproic acid, belinostat, mocetinostat, givinostat, andtrichostatin A. Examples of aurora kinase inhibitors include ZM447439,hesperadin, and VX-680. Examples of demethylating agents include5-azacytidine, 5-azadeoxycytidine, and procaine. Examples ofimmunosuppressive drugs include 6-mercaptopurine, and azathioprine.

Certain Kits

The compounds of the preferred embodiments can be provided to anadministering physician or other health care professional in the form ofa kit. The kit is a package which houses a container which contains thecompounds in a suitable pharmaceutical composition, and instructions foradministering the pharmaceutical composition to a subject. The kit canoptionally also contain one or more additional therapeutic agents, e.g.,chemotherapeutics currently employed for treating the sarcomas describedherein. For example, a kit containing one or more compositionscomprising compounds of the preferred embodiments in combination withone or more additional chemotherapeutic agents can be provided, orseparate pharmaceutical compositions containing an inhibitor of thepreferred embodiments and additional therapeutic agents can be provided.The kit can also contain separate doses of a compound of the preferredembodiments for serial or sequential administration. The kit canoptionally contain one or more diagnostic tools and instructions foruse. The kit can contain suitable delivery devices, e.g., syringes, andthe like, along with instructions for administering the inhibitor(s) andany other therapeutic agent. The kit can optionally contain instructionsfor storage, reconstitution (if applicable), and administration of anyor all therapeutic agents included. The kits can include a plurality ofcontainers reflecting the number of administrations to be given to asubject.

Methods of Use

Some embodiments provided herein relate to methods of treating theEwing's sarcoma family of tumors (ESFT). ESFT contains the unique fusionprotein EWS-FLI1. ESFT affects patients between the ages of 3 and 40years, with most cases occurring in the second decade. Although theembryologic cell type from which ESFT are derived is unknown, the tumoroften grows in close proximity to bone, but can occur as a soft-tissuemass. Over 40% of patients who present with localized tumors willdevelop recurrent disease and the majority of these will die from ESFT,while 75-80% of patients who present with metastatic ESFT will diewithin 5 years despite high-dose chemotherapy (Grier H E, Krailo M D,Tarbell N J, et al. Addition of ifosfamide and etoposide to standardchemotherapy for Ewing's sarcoma and primitive neuroectodermal tumor ofbone. N Engl J Med 2003; 348(8):694-701). These survival rates have notimproved for the past 20 years, even after dose-intensifyingchemotherapy. To improve survival and reduce therapy-related morbidity,novel targeted strategies for treating ESFT patients, as provided in thepreferred embodiments, can be employed.

ESFT are characterized by a translocation, occurring in 95% of tumors,between the central exons of the EWS gene (Ewing Sarcoma) located onchromosome 22 to the central exons of an ets family gene; either FLI1(Friend Leukemia Insertion) located on chromosome 11, t(11;22), or ERGlocated on chromosome 21, t(21;22). The EWS-FLI1 fusion transcriptencodes a 55 kDa protein (electrophoretic motility of approximately 68kD) with two primary domains. The EWS domain is a potent transcriptionalactivator, while the FLI1 domain contains a highly conserved ets DNAbinding domain (May W A, Lessnick S L, Braun B S, et al. The Ewing'ssarcoma EWS/FLI-1 fusion gene encodes a more potent transcriptionalactivator and is a more powerful transforming gene than FLI-1. Mol CellBiol 1993; 13(12):7393-8); the resulting EWS-FLI1 fusion protein acts asan aberrant transcription factor. EWS-FLI1 transformation of mousefibroblasts requires both the EWS and FLI1 functional domains to beintact (May W A, Gishizky M L, Lessnick S L, et al. Ewing sarcoma 11;22translocation produces a chimeric transcription factor that requires theDNA-binding domain encoded by FLI1 for transformation. Proc Natl AcadSci USA 1993; 90(12):5752-6).

EWS-FLI1 is an outstanding therapeutic target, in that it is expressedonly in tumor cells and is required to maintain the growth of ESFT celllines. Reduced expression levels of EWS-FLI1 using either antisenseoligodeoxynucleotides (ODN) (Toretsky J A, Connell Y, Neckers L, Bhat NK. Inhibition of EWS-FLI-1 fusion protein with antisenseoligodeoxynucleotides. J Neurooncol 1997; 31(1-2):9-16; Tanaka K,Iwakuma T, Harimaya K, Sato H, Iwamoto Y. EWS-Fli1 antisenseoligodeoxynucleotide inhibits proliferation of human Ewing's sarcoma andprimitive neuroectodermal tumor cells. J Clin Invest 1997; 99(2):239-47)or small interfering RNAs (siRNA) (Ouchida M, Ohno T, Fujimura Y, Rao VN, Reddy E S. Loss of tumorigenicity of Ewing's sarcoma cells expressingantisense RNA to EWS-fusion transcripts. Oncogene 1995; 11(6):1049-54;Maksimenko A, Malvy C, Lambert G, et al. Oligonucleotides targetedagainst a junction oncogene are made efficient by nanotechnologies.Pharm Res 2003; 20(10):1565-7; Kovar H, Aryee D N, Jug G, et al.EWS/FLI-1 antagonists induce growth inhibition of Ewing tumor cells invitro. Cell Growth Differ 1996; 7(4):429-37) cause decreasedproliferation of ESFT cell lines and regression of tumors in nude mice.Recent advances in nanotechnology have improved the delivery andcontrolled release of siRNA, yet neither antisense ODN nor siRNAreduction of EWS-FLI1 in humans is possible with current technologies(Maksimenko A, Malvy C, Lambert G, et al. Oligonucleotides targetedagainst a junction oncogene are made efficient by nanotechnologies.Pharm Res 2003; 20(10):1565-7; Lambert G, Bertrand J R, Fattal E, et al.EWS FLI-1 antisense nanocapsules inhibits Ewing sarcoma-related tumor inmice. Biochem Biophys Res Commun 2000; 279(2):401-6). One interestingapproach to EWS-FLI1 targeting used comparative expression between siRNAreduced EWS-FLI1 and a library of small molecules, which led to aclinical trial with Ara-C(Stegmaier K, Wong J S, Ross K N, et al.Signature-based small molecule screening identifies cytosine arabinosideas an EWS/FLI modulator in Ewing sarcoma. PLoS medicine 2007;4(4):e122). This method of identifying Ara-C also indicated doxorubicinand puromycin would reduce EWS-FLI1 levels. Doxorubicin is currentlyused as standard therapy for ESFT patients and yet, survival is far fromacceptable (Grier H E, Krailo M D, Tarbell N J, et al. Addition ofifosfamide and etoposide to standard chemotherapy for Ewing's sarcomaand primitive neuroectodermal tumor of bone. N Engl J Med 2003;348(8):694-701). The use of Ara-C in ESFT patients is currently beingevaluated in a Phase II trial. While it is hoped that this represents aneeded clinical breakthrough, it certainly demonstrates the importanceof small molecule targeting of EWS-FLI1. The preferred embodimentsprovide small molecule protein-protein interaction inhibitors (SMPPII)that disrupt EWS-FLI1 from critical protein partners, thereby achievingtumor specificity and more precise targeting of EWS-FLI1.

EWS-FLI1 is a great therapeutic target since it is only expressed intumor cells; however, the ability to target this tumor-specific oncogenehas previously not been successful. One of the challenges towards smallmolecule development is that EWS-FLI1 lacks any known enzymatic domains,and enzyme domains have been thought to be critical for targetedtherapeutics. In addition, EWS-FLI1 is a disordered protein, indicatingthat it does not exhibit a rigid structure that can be used forstructure based drug design (Uren A, Tcherkasskaya O, Toretsky J A.Recombinant EWS-FLI1 oncoprotein activates transcription. Biochemistry2004; 43(42):13579-89). In fact, the disordered nature of EWS-FLI1 iscritical for its transcriptional regulation (Ng K P, Potikyan G, SaveneR O, Denny C T, Uversky V N, Lee K A. Multiple aromatic side chainswithin a disordered structure are critical for transcription andtransforming activity of EWS family oncoproteins. Proc Natl Acad Sci USA2007; 104(2):479-84). Disordered proteins are considered as moreattractive targets for small molecule protein-protein interactioninhibitors specifically because of their biochemical disorderedproperties (Cheng Y, LeGall T, Oldfield C J, et al. Rational drug designvia intrinsically disordered protein. Trends Biotechnol 2006;24(10):435-42)

EWS-FLI1 binds RNA helicase A in vitro and in vivo. It is believed thatprotein-protein interactions of EWS-FLI1 may contribute to its oncogenicpotential; therefore, novel proteins have been sought that directlyinteract with and functionally modulate EWS-FLI1. Recombinant EWS-FLI1that is transcriptionally active (Uren A, Tcherkasskaya 0, Toretsky J A.Recombinant EWS-FLI1 oncoprotein activates transcription. Biochemistry2004; 43(42):13579-89) was used as a target for screening a commercialpeptide phage display library. Twenty-eight novel peptides thatdifferentially bind to EWS-FLI1 were identified from phage sequencing. ANational Center for Biotechnology Information database search for humanproteins homologous to these peptides identified a peptide that washomologous to aa 823-832 of the human RNA helicase A, (RHA, gene bankaccession number A47363) (Toretsky J A, Erkizan V, Levenson A, et al.Oncoprotein EWS-FLI1 activity is enhanced by RNA helicase A. Cancer Res2006; 66(11):5574-81).

While EWS-FLI1 is quite specific to ESFT cells, EWS and RHA areubiquitously expressed. The region between EWS-FLI1 and RHA are targetedby molecular therapeutics that may have specificity; since EWS-FLI1 isexpressed only in tumors and the interaction points with RHA may beunique. Therapeutic agents, namely, small molecule protein-proteininteraction inhibitors, are provided herein to inhibit EWS-FLI1function.

Most translocation-fusion protein sarcomas portend a poor prognosis,including ESFT. The chromosomal translocation t(11;22), leading to theunique and critical fusion protein EWS-FLI1, is a perfect cancer target.Many other sarcomas share similar translocation variants (Table 2. fromHelman L J, Meltzer P. Mechanisms of sarcoma development. Nat Rev Cancer2003; 3(9):685-94).

EWS-FLI1 translocations have been reported in solidpseudopapillaryneoplasms of the pancreas (Maitra A., et al., Detectionof t(11;22)(q24;q12) translocation and EWS-FLI-1 fusion transcript in acase of solid pseudopapillary tumor of the pancreas. Pediatr Dev Pathol2000; 3:603-605), however the role of EWS-FLI1 in all solidpseudopapillary neoplasms remains to be resolved (Katharina Tiemann etal., Solid pseudopapillary neoplasms of the pancreas are associated withFLI-1 expression, but not with EWS/FLI-1 translocation).

EWS or FLI1 homologues are partners in translocations that occur in awide range of sarcomas and leukemias. EWS, or its homologue TLS or FUS,is involved in chromosomal translocations of clear cell sarcoma, myxoidliposarcoma, desmoplastic small round cell tumor, chondrosarcoma andacute myeloid leukemia. FLI1 belongs to the ets family of genes. TheFLI1 homologue ERG is translocated in approximately 10% of Ewing'ssarcomas and 20% of acute myeloid leukemias. This suggests that EWS-FLI1can serve as model system that might impact upon a family of diseases(related by translocation partners) that affect a large number ofpatients (Uren A., Tcherkasskaya O. and Toretsky J. A. RecombinantEWS-FLI1 oncoprotein activates transcription. Biochemistry 43(42)13579-89 (2004)).

ERG is also translocated in prostate cancer, where the TMPRSS2:ERGfusion suggests a distinct molecular subtype that may define risk fordisease progression (F. Demichelis et al., TMPRSS2:ERG gene fusionassociated with lethal cancer in a watchful waiting cohort. Oncogene(2007) 26, 4596-4599). Other diseases where translocations of EWS orFLI1 family members have been observed include prostate cancer,glioblastoma, acute myeloid leukemia, breast cancer, head & neck cancer,melanoma, non-small cell lung cancer, ovarian cancer, and uterine cancer(Janknecht, Ralf; Shin, Sook, and Oh, Sangphil, ETV1, 4 and 5: AnOncogenic Subfamily of ETS Transcription Factors. Biochim. Biophys. Acta1826 (1), 1-12 (2012)).

Therefore, the therapeutic agents of the preferred embodiments havepotential for application in many other tumors. More broadly, some ofthe most difficult leukemias also have translocation-generated fusionproteins involving the mixed-lineage leukemia gene (MLL, 11q23), and ourwork could serve as a paradigm for a very treatment-resistant group ofcancers (Pui C H, Chessells J M, Camitta B, et al. Clinicalheterogeneity in childhood acute lymphoblastic leukemia with 11q23rearrangements. Leukemia 2003; 17(4):700-6). Thus embodiments includecancers where translocations have occurred. Translocation fusion genesare listed in TABLE 1.

TABLE 1 Ewing's sarcoma Translocation Genes Type of fusion gene t(11;22)(q24; q12) EWSR1-FLI1 Transcription factor t(21; 22)(q22; q12)EWSR1-ERG Transcription factor t(7; 22)(p22; q12) EWSR1-ETV1Transcription factor t(17; 22)(q21; q12) EWSR1-ETV4 Transcription factort(2; 22)(q33; q12) EWSR1-FEV Transcription factor

A number of disorders include overexpression of an ETS gene, or an ETSgene fusion, that is, a gene translocation that includes an ETS gene.Examples of such ETS genes include FLI1, ERG, ETV1, and ETV4. Examplesof fusion genes include EWS-FLI, TMPRSS2-ERG. TABLE 1A lists severalcancers in which one or more ETS gene family members are overexpressed,and/or are rearranged.

TABLE 1A Tumors with ETS overexpression or ETS member Cancer gene fusionFLI1 ERG ETV1 ETV4 Prostate 41% 2% 25%  10% 6% Melanoma 34% 8% 8% 20% 5%Non-small-cell lung 33% 12%  8% 12% 5% carcinoma Uterine 25% 6% 9% 11%6% Head and Neck 24% 6% 4%  7% 9% Ovarian 21% 7% 3% 10% 3% Glioblastoma19% 7% 4%  7% 4% multiforme Acute myeloid 19% 8% 8%  4% 2% leukemiaBreast 18% 5% 4%  5% 7%

Indications

Certain compounds, compositions and methods provided herein can be usedto treat a number of disorders such as a tumor or tumor cell comprisinga translocation gene fusion, such as those listed in TABLE 1, Ewing'ssarcoma, prostate cancer, glioblastoma, acute myeloid leukemia, breastcancer, head & neck cancer, melanoma, non-small cell lung cancer,ovarian cancer, and uterine cancer. Some embodiments of the methodsprovided herein include a method for inhibiting proliferation of a cell.In some embodiments, the cell overexpresses an ETS gene. In someembodiments, the overexpressed ETS gene can include FLI1, ERG, ETV1, orETV4. In some embodiments, the cell comprises an ETS fusion gene. Insome embodiments, the ETS fusion gene can include an ETS gene such asFLI1, ERG, ETV1, and ETV4.

EXAMPLES Preparation of Compounds of Formula (I)

Compounds of Formula (I) were prepared according to the syntheticschemes presented herein. A ketone of Formula (II) (4.0 equiv.) wascondensed with an isatin derivative of Formula (III) (1.0 equiv.) in thepresence of diethylamine (10 drops) in methanol (5 mL) and the mixturewas stirred at room temperature for 24 hours. The reaction mixture wasconcentrated and purified using flash chromatography usingdichloromethane/methanol as eluent to yield the pure product. Furtherpurification was done by recrystallization with methanol. Thesubstituents on the ketone and on the isatin derivative were selected soas to yield compounds of Formula (I) labeled as EXAMPLES 1-23 below. NMRSpectra were recorded for the compounds of EXAMPLES 1-26 thus obtainedusing a Varian-400 spectrometer for 1H (400 MHz). Chemical shifts aregiven in ppm downfield from tetramethylsilane as internal standard, andcoupling constants (J-values) are in hertz (Hz).

The chiral separation was done by dissolving the isomeric mixtures inmethanol/methylene chloride (4/1) mixture or neat methanol and theseparation was performed by Supercritical Fluid Chromatography (SFC)using a chiralpak IA column (250 mm×4.6 mm; particle size 5 μm) andeluted using a mixture of methanol/CO₂ (50/50). The solvent was removedunder vacuum to obtain the pure enantiomers.

In some embodiments, compounds can be prepared according to thefollowing synthesis schemes.

In these schemes, ketone (4.0 equiv.) and a catalytic amount ofdiethylamine (10 drops) are added to a solution of substituted isatin(1.0 equiv.) in methanol (5 mL). The mixture is stirred at roomtemperature until starting material (substituted isatin) disappearscompletely. The resulting solution is concentrated and applied to flashchromatography eluting with hexane/ethyl acetate to afford pure productin quantitative yield. Further purification is done by recrystallizationwith hexane/ethyl acetate.

An example compound synthesized by the above scheme includes:4,7-Dichloro-3-hydroxy-3-[2-(4-methoxyphenyl-2-oxoethyl)]-1,3-dihydroindol-2-one:white solid; mp 149-151° C.; ¹H NMR (DMSO, 400 MH 1 z) δ 10.93 (s, 1H),7.86 (d, 2H, J=9.2 Hz), 7.26 (d, 1H, J=8.8 Hz), 6.98 (d, 2H, J=8.8 Hz),6.86 (d, 1H, J=8.4 Hz), 6.39 (s, 1H), 4.31 (d, 1H, J=18.0 Hz), 3.80 (s,3H), 3.61 (d, 1H, J=18.0 Hz).

In some embodiments, compounds can be prepared according to thefollowing synthesis scheme.

In such embodiments, an appropriate acetophenone and 4,7-dichloroisatincan be condensed in the presence of a catalytic amount of diethylamineto prepare the desired compound in quantitative yield. An examplesynthesis includes the following:

4,7-Dichloroisotin (30.05 g, 139.1 mmol, 1.0 equiv, Alfa Aesar lot#10173559) and MeOH (450 mL, 15 vol) were charged to a 2-L, three-neck,round-bottom flask equipped with nitrogen line, overhead mechanicalstirrer, and a temperature probe. Diethylamine (3.25 g, 0.32 equiv,Sigma-Aldrich lot # SHBD5313V) was added over 3 min (the slurry becomesdark red). A very slight increase in temperature (from 17.5° C. to 18.8°C.) was observed. 1-[4-(Dimethylamino)phenyl]ethanone 2 (44.3 g, 1.95equiv, ArkPharm lot #0000197-130717000) was then added via a plasticfunnel and the funnel was rinsed with MeOH (75 mL, 2.5 vol). A decreasein the reaction temperature to 15.1° C. was observed. Upon stirring fora few minutes, a dark red solution with a few undissolved particles wasobtained. The solution was stirred at ambient temperature andperiodically sampled for in-process control (IPC) by HPLC. After 23 h ofreaction, additional diethylamine was added via syringe (1.42 g, 0.14equiv) and the stirring continued at ambient temperature. After 40.5 h,a light slurry formed. Solid 2 was added in portions (54.1 g, 2.38equiv, ArkPharm lot #0000197-130717000 and 3.2 g, 0.14 equiv, TCI lot #GKO1-BRAH) for a total of 4.47 equiv of acetophenone 2. After 88 h ofreaction, IPC by HPLC showed less than 1% AUC of isatin 1 present in thereaction mixture. A heavy precipitate had formed. After 4.5 days, thereaction mixture was concentrated under reduced pressure (water bath<40° C.), then under high vacuum to afford approximately 84 g of a solidmixture, lot # BIO-W-22-11. The solid was dissolved in a mixture ofdichloromethane (385 mL) and MeOH (140 mL) and adsorbed over 100 g ofsilica gel. The solvent was removed under reduced pressure and the dryproduct/silica mixture was loaded onto a column containing silica gel (1kg, pre-packed with heptanes) for a flash chromatographic purification.Elution was started with 10% ethyl acetate in heptanes and a gradient upto 100% ethyl acetate was applied, and then switched to 10% methanol inethyl acetate. Fractions of 500 mL and up to 2 L were collected. Theproduct containing fractions, where product had started to precipitate,were combined and concentrated down to approximately 1 L. The resultingprecipitate was filtered out, reslurried in EtOAc/MeOH (75:25 ratio, 200mL), filtered, and washed with MeOH to afford a first crop of compound.The first filtrate was concentrated to a low volume, added MeOH toprecipitate a second crop of compound. Filtrates from isolation of bothcrops were combined, concentrated to a low volume, taken up in 25 mL ofMeOH, and the resulting solid was filtered to afford a third crop ofcompound. All three crops were dried under high vacuum at ambienttemperature for a day and at 40° C. for four days. The total combinedweight was 40.03 g, corresponding to 76% yield of compound (uncorrectedby purity or solvent content). Solid is off-white (with a very paleyellow to peach shade.

Another example synthesis includes the following:

4,7-Dichloroisotin (4.26 g, 19.7 mmol, 1 equiv, Alfa Aesar lot#10173559) and MeOH (70 mL, 16.4 vol) were charged to a 250-mL,three-neck, round-bottom flask equipped with nitrogen line, overheadmechanical stirrer, and a temperature probe. Diethylamine (0.43 g, 0.30equiv, Sigma-Aldrich lot # SHBD5313V) was added over 1 min via syringe(the slurry becomes dark red). 1-[4-(Methylamino)phenyl]ethanone 3 (11.4g, 3.9 equiv, Sigma-Aldrich lot #01129HHV) was added in portions via aplastic funnel, over 15 min. The funnel was rinsed with MeOH (2×15 mL,7.0 vol). The reaction was stirred at ambient temperature (approximately18-20° C.) and periodically sampled for in-process control (IPC) byHPLC. After 40 h of reaction, additional diethylamine was charged to thereaction via syringe (0.16 g, 0.11 equiv) and the stirring continued atambient temperature. After 64 h, a light slurry formed. Additionaldiethylamine was charged to the reaction via syringe (0.13 g, 0.09equiv) and the stirring continued at ambient temperature. After 92 h ofreaction IPC by HPLC analysis showed 2.1% AUC of isatin 1 present in thereaction mixture. Additional diethylamine was charged to the reactionvia syringe (0.07 g, 0.05 equiv) for a total of 0.55 equiv of base andthe stirring continued at ambient temperature over the weekend. After atotal of seven days, the reaction was concentrated under reducedpressure (water bath <40° C.), the solid residue was dissolved in amixture of dichloromethane (450 mL) and MeOH (50 mL) at 30° C., andadsorbed over 20 g of silica gel. Purification was carried out in aCombiflash Companion™ XL system with a RediSep disposable flash 220 gsilica gel column (catalog #69-2203-422). Elution of the residualstarting material 3 was accomplished with dichloromethane (approximately20 column vol, while the product TK-202 was eluted with 10% methanol indichloromethane. The product containing fractions, where product hadalready started to precipitate, were combined in two different lots andpartially concentrated under reduced pressure. The resulting slurrieswere filtered and the solid cakes were washed with MeOH to afford twofractions that were dried under high vacuum at ambient temperature for24 h, then 50° C. for 24 h, to afford lot # BIO-W-30-17 and lot #BIO-W-30-18. The filtrate from both crystallizations were combined andsubjected to a second chromatographic purification (on a 40-g RediSepGold column, catalog #69-2203-347) using a gradient from dichloromethaneto 5% methanol in dichloromethane for elution. The product containingfractions (purity higher than 99% AUC by HPLC) were combined and theproduct was allowed to precipitate over 2 h. The solid was filtered off,washed with methanol, and dried under high vacuum for 24 h at 50° C. toafford lot # BIOW-30-19. A second set of fractions containing product ofapproximately 95% AUC purity by HPLC were combined, the solid wasfiltered off, re-dissolved in dichloromethane, and added 20% methanol toprecipitate overnight. The precipitated TK-202 was filtered and washedwith methanol, then dried under high vacuum for 24 h at 50° C. to affordlot # BIO-W-30-16. The total combined weight of 5.99 g corresponds to83% yield of compound. Solid is off-white (with a very pale peach to tanshade).

4,7-Dichloro-3-hydroxy-3-(2-(4-(methylsulfonyl)phenyl)-2-oxoethyl)indolin-2-one(Example 1): off-white solid; ¹H NMR (DMSO-d6, 400 MHz) δ 3.28 (s, 3H),3.81 (d, 1H, J=16 Hz), 4.42 (d, 1H, J=16 Hz), 6.53 (s, 1H), 6.92 (d, 1H,J=8 Hz), 7.32 (d, 1H, J=8 Hz), 8.05 (d, 2H, J=8 Hz), 8.17 (d, 2H, J=8Hz), 11.04 (s, 1H).

3-(2-(4-(Aziridin-1-yl)phenyl)-2-oxoethyl)-4,7-dichloro-3-hydroxyindolin-2-one(Example 2):

To 4,7-dichloroindoline-2,3-dione (A) (300 mg, 1.39 mmol) in 15 mL ofmethanol were 1-(4-(aziridin-1-yl)phenyl)ethanone (B) (0.9 g, 5.5 mmol)and 10 drops of diethylamine (2). The reaction was stirred at 50° C. for24 hours. The solvent was removed and the residue was purified withflash chromatography (0-5% Methanol/CH2Cl2) to get an off white solid.3-(2-(4-(Aziridin-1-yl)phenyl)-2-oxoethyl)-4,7-dichloro-3-hydroxyindolin-2-one(Example 2): off-white solid; ¹H NMR (DMSO-d6, 400 MHz) δ 2.16 (s, 4H),3.64 (d, 1H, J=16 Hz), 4.32 (d, 1H, J=16 Hz), 6.41 (s, 1H), 6.89 (d, 1H,J=8 Hz), 7.05 (d, 2H, J=8 Hz), 7.30 (d, 1H, J=8 Hz), 7.80 (d, 2H, J=8Hz), 10.95 (s, 1H).

4,7-Dichloro-3-hydroxy-3-(2-(4-isopropylphenyl)-2-oxoethyl)indolin-2-one(Example 3): off-white solid; ¹H NMR (DMSO-d6, 400 MHz) δ 1.21 (d, 6H,J=4 Hz), 2.95 (m, 1H), 3.69 (d, 1H, J=16 Hz), 4.39 (d, 1H, J=16 Hz),6.45 (s, 1H), 6.90 (d, 1H, J=8 Hz), 7.29 (d, 1H, J=8 Hz), 7.38 (d, 2H,J=8 Hz), 7.85 (d, 2H, J=8 Hz), 10.98 (s, 1H).

4,7-Dichloro-3-(1-(4-(dimethylamino)phenyl)-1-oxopropan-2-yl)-3-hydroxyindolin-2-one(Example 4): ¹H NMR (DMSO-d6, 400 MHz) δ 1.48 (d, 3H, J=8 Hz), 3.00 (s,6H), 4.78 (m, 1H), 6.35 (s, 1H), 6.66 (d, 2H, J=8 Hz), 6.76 (d, 1H, J=8Hz), 7.17 (d, 1H, J=8 Hz), 7.69 (d, 2H, J=8 Hz), 10.74 (s, 1H).

4,7-Dichloro-3-(2-(4-cyclopropylphenyl)-2-oxoethyl)-3-hydroxyindolin-2-one(Example 7):

To 4,7-dichloroindoline-2,3-dione (A) (300 mg, 1.39 mmol) in 15 mL ofmethanol were added 1-(4-cyclopropylphenyl)ethanone (B) (0.9 g, 5.5mmol) and 10 drops of diethylamine (2). The reaction was stirred at 50°C. for 24 hours. The solvent was removed and the residue was purifiedwith flash chromatography (0-5% Methanol/CH2Cl2) to get an off whitesolid.4,7-Dichloro-3-(2-(4-cyclopropylphenyl)-2-oxoethyl)-3-hydroxyindolin-2-one(Example 7): off-white solid; ¹H NMR (DMSO-d6, 400 MHz) δ 0.76 (m, 2H),1.06 (m, 2H), 2.0 (m, 1H), 3.65 (d, 1H, J=16 Hz), 4.35 (d, 1H, J=16 Hz),6.43 (s, 1H), 6.89 (d, 1H, J=8 Hz), 7.19 (d, 2H, J=8 Hz), 7.30 (d, 1H,J=8 Hz), 7.79 (d, 2H, J=8 Hz), 10.97 (s, 1H).

3-(2-(4-(1H-Pyrazol-1-yl)phenyl)-2-oxoethyl)-4,7-dichloro-3-hydroxyindolin-2-one(Example 8): off-white solid; ¹H NMR (DMSO-d6, 400 MHz) δ 3.72 (d, 1H,J=16 Hz), 4.41 (d, 1H, J=16 Hz), 6.47 (s, 1H), 6.62 (d, 1H, J=4 Hz),6.90 (d, 1H, J=8 Hz), 7.31 (d, 1H, J=8 Hz), 7.84, (d, 1H, J=4H), 7.99(d, 2H, J=8 Hz), 8.06 (d, 2H, J=8 Hz), 8.66 (d, 1H, J=4 Hz), 10.98 (s,1H).

4,7-Dichloro-3-hydroxy-3-(2-oxo-2-(4-(pyrrolidin-1-yl)phenyl)ethyl)indolin-2-one(Example 10):

To 4,7-dichloroindoline-2,3-dione (A) (12.5 g, 0.06 mol) in 800 mL ofmethanol were added 1-(4-(pyrrolidin-1-yl)phenyl)ethanone (B) (45 g,0.24 mol) and 0.5 mL of diethylamine (2). The reaction was stirred at rtfor 24 hours. The solvent was removed and the residue was purified withflash chromatography (0-5% Methanol/CH₂C₁₂) to get 13.5 g of brownsolid. It was purified again with flash chromatography using ethylacetate/hexane to give 11.5 g of an off white solid. Repeating thereaction at the same scale to give another 11.5 g of product. Twobatches of product were combined and recrystallized from methanol to get20.5 g as an off white solid.4,7-Dichloro-3-hydroxy-3-(2-oxo-2-(4-(pyrrolidin-1-yl)phenyl)ethyl)indolin-2-one(Example 10): off-white solid; ¹H NMR (DMSO-d6, 400 MHz) δ 1.96 (m, 4H),3.30 (m, 4H), 3.65 (d, 1H, J=16 Hz), 4.29 (d, 1H, J=16 Hz), 6.34 (s,1H), 6.53 (d, 2H, J=8 Hz), 6.88 (d, 1H, J=8 Hz), 7.28 (d, 1H, J=8 Hz),7.72 (d, 2H, J=8 Hz), 10.97 (s, 1H). Chiral separation was performed onunder the following conditions. Preparatory method utilized thefollowing: a RegisCell column L: 250 mm, IS: 50 mm, particle size: 5 μm;mobile phase: methanol/CO₂, ratio: 35/65, detection wavelength: 254 nm,flow rate: 325 g/min, co-solvent flow rate 113.75 ml/min. Dissolved19.72 g in 1000 ml of methanol, for a concentration of 0.020 g/ml. Theinjection volume was 25.00 ml for a total amount 0.500 g/injection.Yield was (+): 9.73 g, with optical rotation +247 at 20° C. and (−):9.26 g.

4-(2-(4,7-Dichloro-3-hydroxy-2-oxoindolin-3-yl)acetyl)benzenesulfonamide(Example 11): off-white solid; ¹H NMR (DMSO-d6, 400 MHz) δ 3.78 (d, 1H,J=16 Hz), 4.41 (d, 1H, J=16 Hz), 6.51 (s, 1H), 6.90 (d, 1H, J=8 Hz),7.31 (d, 1H, J=8 Hz), 7.56 (s, 2H), 7.91 (d, 2H, J=8 Hz), 8.11 (d, 2H,J=8 Hz), 10.98 (s, 1H).

4,7-Dichloro-3-(2-(3-fluoro-4-(pyrrolidin-1-yl)phenyl)-2-oxoethyl)-3-hydroxyindolin-2-one(Example 12): off-white solid; ¹H NMR (DMSO-d6, 400 MHz) δ 1.91 (m, 4H),3.46 (m, 4H), 3.57 (d, 1H, J=16 Hz), 4.27 (d, 1H, J=16 Hz), 6.36 (s,1H), 6.71 (t, 1H, J=4 Hz, J=8 Hz), 7.29 (d, 1H, J=8 Hz), 7.46 (d, 1H,J=8 Hz), 7.61 (d, 1H, J=4 Hz), 7.64 (d, 1H, J=8 Hz), 11.01 (s, 1H).

3-(2-(4-(Azetidin-1-yl)phenyl)-2-oxoethyl)-4,7-dichloro-3-hydroxyindolin-2-one(Example 13):

To 4,7-dichloroindoline-2,3-dione (A) (300 mg, 1.39 mmol) in 15 mL ofmethanol were added 1-(4-(azetidin-1-yl)phenyl)ethan-1-one (B) (972 mg,5.5 mmol) and a few drops of diethylamine (2). The reaction was stirredat rt for 24 hours. The solvent was removed and the residue was purifiedwith flash chromatography (0-5% Methanol/CH2Cl2) to get an off whitesolid.3-(2-(4-(Azetidin-1-yl)phenyl)-2-oxoethyl)-4,7-dichloro-3-hydroxyindolin-2-one(Example 13): off-white solid; ¹H NMR (DMSO-d6, 400 MHz) δ 2.32 (m, 2H),3.51 (d, 1H, J=16 Hz), 3.95 (m, 4H), 4.30 (d, 1H, J=16 Hz), 6.35 (s,1H), 6.36 (d, 2H, J=8 Hz), 6.87 (d, 1H, J=8 Hz), 7.28 (d, 1H, J=8 Hz),7.73 (d, 2H, J=8 Hz), 10.89 (s, 1H).

4,7-Dichloro-3-hydroxy-3-(2-(4-methoxycyclohexyl)-2-oxoethyl)indolin-2-one(Example 14): off-white solid; ¹H NMR (CDCl₃, 400 MHz) δ 1.24 (m, 4H),1.92 (m, 2H), 2.08 (m, 2H), 2.32 (m, 1H), 3.06 (m, 1H), 3.26 (s, 3H),3.33 (d, 1H, J=16 Hz), 3.69 (s, 1H), 3.70 (d, 1H, J=16 Hz), 6.91 (d, 1H,J=8 Hz), 7.20 (d, 1H, J=8 Hz), 7.61 (s, 1H).

4,7-Dichloro-3-hydroxy-3-(2-(4-methoxybicyclo[2.2.2]octan-1-yl)-2-oxoethyl)indolin-2-one(Example 15): off-white solid; ¹H NMR (CDCl₃, 400 MHz) δ 1.59 (m, 12H),3.16 (s, 3H), 3.22 (d, 1H, J=16 Hz), 3.58 (s, 1H), 4.14 (d, 1H, J=16Hz), 6.90 (d, 1H, J=8 Hz), 7.23 (d, 1H, J=8 Hz), 7.67 (s, 1H).

4,7-Dichloro-3-(2-(4-(dimethylamino)bicyclo[2.2.2]octan-1-yl)-2-oxoethyl)-3-hydroxyindolin-2-one(Example 16): off-white solid; ¹H NMR (DMSO-d6, 400 MHz) δ 1.59 (m,12H), 2.5 (s, 3H), 3.18 (d, 1H, J=16 Hz), 3.85 (d, 1H, J=16 Hz), 6.31(s, 1H), 6.90 (d, 1H, J=8 Hz), 7.30 (d, 1H, J=8 Hz), 10.95 (s, 1H).

4,7-Dichloro-3-(2-(4-cyclopropyl-3-fluorophenyl)-2-oxoethyl)-3-hydroxyindolin-2-one(Example 17):

To 4,7-dichloroindoline-2,3-dione (A) (261 mg, 1.21 mmol) in 15 mL ofmethanol were 1-(4-cyclopropyl-3-fluorophenyl)ethanone (B) (280 mg, 1.57mmol) and 10 drops of diethylamine (2). The reaction was stirred at 50°C. for 24 hours. The solvent was removed and the residue was purifiedwith flash chromatography (0-5% Methanol/CH2Cl2) to get an off whitesolid.4,7-Dichloro-3-(2-(4-cyclopropyl-3-fluorophenyl)-2-oxoethyl)-3-hydroxyindolin-2-one(Example 17): off-white solid; ¹H NMR (DMSO-d6, 400 MHz) δ 0.83 (m, 2H),1.08 (m, 2H), 2.11 (m, 1H), 3.68 (d, 1H, J=16 Hz), 4.34 (d, 1H, J=16Hz), 6.43 (s, 1H), 6.90 (d, 1H, J=8 Hz), 7.11 (m, 1H), 7.30 (d, 1H, J=8Hz), 7.60 (d, 1H, J=8 Hz), 7.68 (d, 1H, J=8 Hz), 10.96 (s, 1H). Chiralseparation was performed by a method substantially similar to the methoddescribed above. LC screening was performed with: column: AD-H, 250mm×4.6 mm, 5 μm, hexane/ethanol (65/35), 1.5 ml/min, injection volume:10.0 μl, pressure: 102.9 bar. Peak 1: retention time: 5.40 min, width:0.171 min, area: 4502.21, area %: 50.08. Peak 2: retention time: 7.23min, width: 0.239 min, area: 4488.43, area %: 49.92.

4,7-Dichloro-3-(2-(4-cyclopropyl-2-fluorophenyl)-2-oxoethyl)-3-hydroxyindolin-2-one(Example 18):

To 4,7-dichloroindoline-2,3-dione (A) (261 mg, 1.21 mmol) in 15 mL ofmethanol were 1-(4-cyclopropyl-2-fluorophenyl)ethanone (B) (280 mg, 1.57mmol) and 10 drops of diethylamine (2). The reaction was stirred at 50°C. for 24 hours. The solvent was removed and the residue was purifiedwith flash chromatography (0-5% Methanol/CH2Cl2) to get an off whitesolid.4,7-Dichloro-3-(2-(4-cyclopropyl-2-fluorophenyl)-2-oxoethyl)-3-hydroxyindolin-2-one(Example 18): off-white solid; ¹H NMR (DMSO-d6, 400 MHz) δ 0.82 (m, 2H),1.07 (m, 2H), 2.01 (m, 1H), 3.66 (d, 1H, J=16 Hz), 4.26 (d, 1H, J=16Hz), 6.44 (s, 1H), 6.91 (d, 1H, J=8 Hz), 7.01 (m, 2H), 7.31 (d, 1H, J=8Hz), 7.57 (m, 1H), 10.96 (s, 1H). Chiral separation was performed by amethod substantially similar to the method described above. LC screeningwas performed with: column: RegisCell, 250 mm×4.6 mm, 5 μm, hexane/IPA(80/20), 1.5 ml/min, injection volume: 2.0 pressure: 51.5 bar. Peak 1:retention time: 5.16 min, width: 0.238 min, area: 3716.20, area %:49.78. Peak 2: retention time: 6.49 min, width: 0.324 min, area:3749.55, area %: 50.22.

4-Chloro-7-fluoro-3-hydroxy-3-(2-(4-methoxyphenyl)-2-oxoethyl)indolin-2-one(Example 19): off-white solid; ¹H NMR (DMSO-d6, 400 MHz) δ 3.63 (d, 1H,J=16 Hz), 3.84 (s, 3H), 4.36 (d, 1H, J=16 Hz), 6.38 (s, 1H), 6.88 (d,1H, J=8 Hz), 7.02 (d, 2H, J=8 Hz), 7.16 (m, 1H), 7.89 (d, 2H, J=8 Hz),11.01 (s, 1H).

4,7-dichloro-3-hydroxy-3-(2-(4-morpholinophenyl)-2-oxoethyl)indolin-2-one(Example 20): off-white solid; ¹H NMR (DMSO-d6, 400 MHz) δ 3.28 (m, 4H),3.53 (d, 1H, J=16 Hz), 3.71 (m, 4H), 4.33 (d, 1H, J=16 Hz), 6.37 (s,1H), 6.87 (d, 1H, J=8 Hz), 6.97 (d, 2H, J=8 Hz), 7.30 (d, 1H, J=8 Hz),7.77 (d, 2H, J=8 Hz), 10.95 (s, 1H).

4,7-dichloro-3-hydroxy-3-(2-oxo-2-(pyridin-4-yl)ethyl)indolin-2-one(EXAMPLE 21): off-white solid; ¹H NMR (DMSO-d6, 400 MHz) δ 3.78 (d, 1H,J=16 Hz), 4.39 (d, 1H, J=16 Hz), 6.53 (s, 1H), 6.92 (d, 1H, J=8 Hz),7.32 (d, 1H, J=8 Hz), 7.79 (d, 2H, J=4 Hz), 8.80 (d, 2H, J=4 Hz), 11.03(s, 1H).

4,7-dichloro-3-hydroxy-3-(2-oxo-2-(pyridin-3-yl)ethyl)indolin-2-one(EXAMPLE 22): off-white solid; ¹H NMR (DMSO-d6, 400 MHz) δ 3.78 (d, 1H,J=16 Hz), 4.39 (d, 1H, J=16 Hz), 6.50 (s, 1H), 6.90 (d, 1H, J=8 Hz),7.30 (d, 1H, J=8 Hz), 7.57 (m, 1H), 8.28 (d, 1H, J=4 Hz), 8.80 (d, 1H,J=4 Hz), 9.09 (s, 1H), 11.03 (s, 1H).

4,7-dichloro-3-hydroxy-3-(2-oxo-2-(pyridin-2-yl)ethyl)indolin-2-one(EXAMPLE 23): off-white solid; ¹H NMR (DMSO-d6, 400 MHz) δ 3.78 (d, 1H,J=16 Hz), 4.68 (d, 1H, J=16 Hz), 6.50 (s, 1H), 6.90 (d, 1H, J=8 Hz),7.30 (d, 1H, J=8 Hz), 7.70 (d, 1H, J=4 Hz), 7.81 (d, 1H, J=4 Hz), 7.98(m, 1H), 8.76 (s, 1H), 11.01 (s, 1H).

4-(2-(4,7-dichloro-3-hydroxy-2-oxoindolin-3-yl)acetyl)benzamide (Example24): off-white solid; ¹H NMR (DMSO-d6, 400 MHz) δ 3.74 (d, 1H, J=16 Hz),4.42 (d, 1H, J=16 Hz), 6.49 (s, 1H), 6.92 (d, 1H, J=8 Hz), 7.31 (d, 1H,J=8 Hz), 7.57 (s, 1H), 7.95 (d, 2H, J=8 Hz), 7.97 (d, 2H, J=8 Hz), 8.10(s, 1H), 11.01 (s, 1H).

4,7-dichloro-3-hydroxy-3-(2-(4-hydroxyphenyl)-2-oxoethyl)indolin-2-one(Example 25): off-white solid; ¹H NMR (DMSO-d6, 400 MHz) δ 3.60 (d, 1H,J=16 Hz), 4.30 (d, 1H, J=16 Hz), 6.39 (s, 1H), 6.82 (d, 2H, J=8 Hz),6.90 (d, 1H, J=8 Hz), 7.30 (d, 1H, J=8 Hz), 7.80 (d, 2H, J=8 Hz), 10.45(s, 1H), 10.93 (s, 1H).

4,7-dichloro-3-(2-(3,4-difluorophenyl)-2-oxoethyl)-3-hydroxyindolin-2-one(Example 26): off-white solid; ¹H NMR (DMSO-d6, 400 MHz) δ 3.70 (d, 1H,J=16 Hz), 4.37 (d, 1H, J=16 Hz), 6.48 (s, 1H), 6.91 (d, 1H, J=8 Hz),7.30 (d, 1H, J=8 Hz), 7.56 (m, 1H), 7.96 (m, 1H), 8.01 (m, 1H), 11.01(s, 1H).

Biological Activity of Compounds

Biological activities of certain compounds listed in TABLE 2 weredetermined.

TABLE 2 Compound (optical rotation) Structure Name  1 (+/−)

4,7-Dichloro-3- hydroxy-3-[2-(4- methoxyphenyl- 2-oxoethyl)]-1,3-dihydroindol-2- one  2 (−)

4,7-Dichloro-3- hydroxy-3-[2-(4- methoxyphenyl- 2-oxoethyl)]-1,3-dihydroindol-2- one  3 (+)

4,7-Dichloro-3- hydroxy-3-[2-(4- methoxyphenyl- 2-oxoethyl)]- 1,3-dihydroindol-2- one  4 (+/−)

4,7-Dichloro-3- hydroxy-3-(2-(4- (methylsulfonyl) phenyl)-2-oxoethyl)indolin- 2-one  5 (−)

4,7-dichloro-3- hydroxy-3-[2-[4- (methylamino) phenyl]-2- oxoethyl]-1H-indol-2-one  6 (+)

4,7-dichloro-3- hydroxy-3-[2-[4- (methylamino) phenyl]-2- oxoethyl]-1H-indol-2-one  7 (+/−)

4,7-dichloro-3- hydroxy-3-[2-[4- (methylamino) phenyl]-2- oxoethyl]-1H-indol-2-one  8 (+/−)

3-(2-(4-(Aziridin- 1-yl)phenyl)-2- oxoethyl)-4,7- dichloro-3-hydroxyindolin- 2-one  9 (+/−)

4,7-Dichloro-3- hydroxy-3-(2- (4-isopropyl- phenyl)- 2-oxoethyl)indolin-2-one 10 (+)

4,7-Dichloro-3- (1-(4- (dimethylamino) phenyl)-1- oxopropan-2-yl)- 3-hydroxyindolin- 2-one 11 (−)

4,7-Dichloro-3- (1-(4- (dimethylamino) phenyl)-1- oxopropan-2-yl)- 3-hydroxyindolin- 2-one 12 (+/−)

4,7-Dichloro-3- (1-(4- (dimethylamino) phenyl)-1- oxopropan-2-yl)- 3-hydroxyindolin- 2-one 13 (+)

4,7-Dichloro-3- (2-(4-cyclo- propylphenyl)- 2-oxoethyl)-3-hydroxyindolin- 2-one 14 (−)

4,7-Dichloro-3- (2-(4-cyclo- propylphenyl)- 2-oxoethyl)-3-hydroxyindolin- 2-one 15 (+/−)

4,7-Dichloro-3- (2-(4-cyclo- propylphenyl)- 2-oxoethyl)-3-hydroxyindolin- 2-one 16 (+/−)

3-(2-(4-(1H- Pyrazol-1- yl)phenyl)- 2-oxoethyl)-4,7- dichloro-3-hydroxyindolin- 2-one 17 (+)

4,7-Dichloro-3- hydroxy-3-(2- oxo-2- (4-(pyrrolidin-1- yl)phenyl)ethyl)indolin-2-one 18 (−)

4,7-Dichloro-3- hydroxy-3-(2- oxo-2-(4- (pyrrolidin-1- yl)phenyl)ethyl)indolin-2-one 19 (+/−)

4,7-Dichloro-3- hydroxy-3-(2- oxo-2-(4- (pyrrolidin-1- yl)phenyl)ethyl)indolin-2-one 20 (+/−)

4-(2-(4,7- Dichloro- 3-hydroxy-2- oxoindolin-3- yl)acetyl) benzene-sulfonamide 21 (+/−)

4,7-Dichloro-3- (2-(3-fluoro-4- (pyrrolidin-1- yl)phenyl)-2-oxoethyl)-3- hydroxyindolin- 2-one 22 (+/−)

3-(2-(4-(Azetidin- 1-yl)phenyl)-2- oxoethyl)-4,7- dichloro-3-hydroxyindolin- 2-one 23 (+/−)

4,7-Dichloro-3- hydroxy-3-(2-(4- methoxy- cyclohexyl)-2-oxoethyl)indolin- 2-one 24 (+/−)

4,7-Dichloro-3- hydroxy-3-(2-(4- methoxybicyclo [2.2.2]octan-1-yl)-2-oxoethyl) indolin-2-one 25 (+/−)

4,7-Dichloro-3- (2-(4- (dimethylamino) bicyclo[2.2.2] octan-1-yl)-2-oxoethyl)-3- hydroxyindolin- 2-one 26 (+/−)

4,7-Dichloro-3- (2-(4- cyclopropyl-3- fluorophenyl)-2- oxoethyl)-3-hydroxyindolin- 2-one 27 (+/−)

4,7-Dichloro-3- (2-(4- cyclopropyl-2- fluorophenyl)-2- oxoethyl)-3-hydroxyindolin- 2-one 27 (−)

4,7-Dichloro-3- (2-(4- cyclopropyl-2- fluorophenyl)-2- oxoethyl)-3-hydroxyindolin- 2-one 27 (+)

4,7-Dichloro-3- (2-(4- cyclopropyl-2- fluorophenyl)-2- oxoethyl)-3-hydroxyindolin- 2-one 28 (+/−)

4-Chloro-7- fluoro-3- hydroxy-3-(2-(4- methoxyphenyl)- 2-oxoethyl)indolin-2-one 29 (+/−)

4,7-dichloro-3- hydroxy-3-(2-(4- morpholino- phenyl)-2-oxoethyl)indolin- 2-one 30 (+/−)

4,7-dichloro-3- hydroxy-3-(2- oxo-2-(pyridin- 4-yl)ethyl) indolin-2-one31 (+/−)

4,7-dichloro-3- hydroxy-3-(2- oxo-2-(pyridin- 3-yl)ethyl) indolin-2-one32 (+/−)

4,7-dichloro-3- hydroxy-3-(2- oxo-2-(pyridin- 2-yl)ethyl) indolin-2-one33 (+/−)

4-(2-(4,7-dichloro- 3-hydroxy-2- oxoindolin-3-yl) acetyl) benzamide 34(+/−)

4,7-dichloro-3- hydroxy-3-(2-(4- hydroxyphenyl)- 2-oxoethyl)indolin-2-one 35 (+/−)

4,7-dichloro-3- (2-(3,4- difluorophenyl)- 2-oxoethyl)-3- hydroxyindolin-2-one 36 (−)

4,7-Dichloro-3- (2-(4- cyclopropyl-3- fluorophenyl)-2- oxoethyl)-3-hydroxyindolin- 2-one 37 (+)

4,7-Dichloro-3- (2-(4- cyclopropyl-3- fluorophenyl)-2- oxoethyl)-3-hydroxyindolin- 2-one 38 (−)

4,7-Dichloro-3- (2-(4- cyclopropyl-2- fluorophenyl)-2- oxoethyl)-3-hydroxyindolin- 2-one 39 (+)

4,7-Dichloro-3- (2-(4- cyclopropyl-2- fluorophenyl)-2- oxoethyl)-3-hydroxyindolin- 2-one

Activities of Compounds

A modified tetrazolium salt assay using the CCK-8 kit (Sigma-Aldrich; StLouis, Mo.) was used to measure the inhibition of human tumor cellgrowth. Tumor cells (5000-7500 per well) were added to 96 well platesand allowed to attach for 4-5 hours. Compounds were serially diluted andadded in triplicate at a concentration of 0.02 to 5 μM. DMSO wasincluded as a vehicle control. Cells were incubated in the presence ofcompound for 3 days. After incubation CCK-8 reagent was added to eachwell and incubated for 2-4 hours. Viable cells were quantitatedspectrophotometrically at a wavelength of 450 nm. Percent viability ofeach sample was calculated from the A450 values as follows: %viability=(A450 nm sample/A450 nm DMSO-treated cells×100). The IC₅₀ wasdefined as the concentration that gave rise to 50% inhibition of cellviability. IC₅₀ activities of particular compounds were determined usingSKES cells (Ewing Sarcoma cell line). Results are summaries in TABLE 3.IC₅₀ activities of particular compounds were determined using the celllines listed in TABLE 4.

TABLE 3 Cmpd IC₅₀ 1 B 2 B 3 A 4 A 5 C 6 C 7 C 8 B 9 A 10 A 11 B 12 C 13A 14 B 15 C 16 A 17 A 18 B 19 C 20 A 21 A 22 B 23 A 24 A 25 A 26 B 27 B28 A 29 A 30 A 31 A 32 A 33 A 34 A 35 A 36 C 37 C 38 C 39 C A is IC₅₀ >5 μM; B is IC₅₀ < 5 μM; and C is not determined.

TABLE 4 Cell Line Tumor Type ETS family rearrangement SKES ES EWS-FLI1Type 2 A4573 ES EWS-FLI1 Type 3 TC71 ES EWS-FLI1 Type 1 LNCap Prostaterearranged ETV1 PC3 Prostate none MDA-MB-231 Breast increased ETV1 MCF7Breast none BxPC3 Pancreas increased FLI1 PANC1 Pancreas none

Results for IC₅₀ of certain compounds are summarized as follows: Cmpd 1:SKES: C A4573: C; TC71: C; LNCap: C; PC3: C; MDA-MB-231: C; MCF7: C;BxPC3: C; and PANC1: C. Cmpd 2: SKES: B; A4573: B; TC71: B; LNCap: B;PC3: A; MDA-MB-231: B; MCF7: A; BxPC3: B; and PANC1: A. Cmpd 3: SKES: C;A4573: C; TC71: C; LNCap: C; PC3: C; MDA-MB-231: C; MCF7: C; BxPC3: C;and PANC1: C. Cmpd 4: SKES: A; A4573: C; TC71: C; LNCap: C; PC3: C;MDA-MB-231: C; MCF7: C; BxPC3: C; and PANC1: C. Cmpd 5: SKES: C; A4573:C; TC71: C; LNCap: C; PC3: C; MDA-MB-231: C; MCF7: C; BxPC3: C; andPANC1: C. Cmpd 6: SKES: C; A4573: C; TC71: C; LNCap: C; PC3: C;MDA-MB-231: C; MCF7: C; BxPC3: C; and PANC1: C. Cmpd 7: SKES: C; A4573:C; TC71: C; LNCap: C; PC3: C; MDA-MB-231: C; MCF7: C; BxPC3: C; andPANC1: C. Cmpd 8: SKES: B; A4573: C; TC71: C; LNCap: C; PC3: C;MDA-MB-231: C; MCF7: C; BxPC3: C; and PANC1: C. Cmpd 9: SKES: A; A4573:C; TC71: C; LNCap: C; PC3: C; MDA-MB-231: C; MCF7: C; BxPC3: C; andPANC1: C. Cmpd 10: SKES: A; A4573: C; TC71: C; LNCap: C; PC3: C;MDA-MB-231: C; MCF7: C; BxPC3: C; and PANC1: C. Cmpd 11: SKES: B; A4573:C; TC71: C; LNCap: C; PC3: C; MDA-MB-231: C; MCF7: C; BxPC3: C; andPANC1: C. Cmpd 12: SKES: C; A4573: C; TC71: C; LNCap: C; PC3: C;MDA-MB-231: C; MCF7: C; BxPC3: C; and PANC1: C. Cmpd 13: SKES: A; A4573:C; TC71: C; LNCap: C; PC3: C; MDA-MB-231: C; MCF7: C; BxPC3: C; andPANC1: C. Cmpd 14: SKES: B; A4573: B; TC71: B; LNCap: B; PC3: A;MDA-MB-231: B; MCF7: A; BxPC3: B; and PANC1: A. Cmpd 15: SKES: C; A4573:C; TC71: C; LNCap: C; PC3: C; MDA-MB-231: C; MCF7: C; BxPC3: C; andPANC1: C. Cmpd 16: SKES: A; A4573: C; TC71: C; LNCap: C; PC3: C;MDA-MB-231: C; MCF7: C; BxPC3: C; and PANC1: C. Cmpd 17: SKES: A; A4573:C; TC71: C; LNCap: C; PC3: C; MDA-MB-231: C; MCF7: C; BxPC3: C; andPANC1: C. Cmpd 18: SKES: B; A4573: C; TC71: C; LNCap: C; PC3: C;MDA-MB-231: C; MCF7: C; BxPC3: C; and PANC1: C. Cmpd 19: SKES: C; A4573:C; TC71: C; LNCap: C; PC3: C; MDA-MB-231: C; MCF7: C; BxPC3: C; andPANC1: C. Cmpd 20: SKES: A; A4573: C; TC71: C; LNCap: C; PC3: C;MDA-MB-231: C; MCF7: C; BxPC3: C; and PANC1: C. Cmpd 21: SKES: A; A4573:C; TC71: C; LNCap: C; PC3: C; MDA-MB-231: C; MCF7: C; BxPC3: C; andPANC1: C. Cmpd 22: SKES: B; A4573: C; TC71: C; LNCap: C; PC3: C;MDA-MB-231: C; MCF7: C; BxPC3: C; and PANC1: C. Cmpd 23: SKES: A; A4573:C; TC71: C; LNCap: C; PC3: C; MDA-MB-231: C; MCF7: C; BxPC3: C; andPANC1: C. Cmpd 24: SKES: A; A4573: C; TC71: C; LNCap: C; PC3: C;MDA-MB-231: C; MCF7: C; BxPC3: C; and PANC1: C. Cmpd 25: SKES: A; A4573:C; TC71: C; LNCap: C; PC3: C; MDA-MB-231: C; MCF7: C; BxPC3: C; andPANC1: C. Cmpd 26: SKES: B; A4573: C; TC71: C; LNCap: C; PC3: C;MDA-MB-231: C; MCF7: C; BxPC3: C; and PANC1: C. Cmpd 27: SKES: B; A4573:C; TC71: C; LNCap: C; PC3: C; MDA-MB-231: C; MCF7: C; BxPC3: C; andPANC1: C. Cmpd 28: SKES: A; A4573: C; TC71: C; LNCap: C; PC3: C;MDA-MB-231: C; MCF7: C; BxPC3: C; and PANC1: C. Cmpd 29: SKES: A; A4573:C; TC71: C; LNCap: C; PC3: C; MDA-MB-231: C; MCF7: C; BxPC3: C; andPANC1: C. Cmpd 30: SKES: A; A4573: C; TC71: C; LNCap: C; PC3: C;MDA-MB-231: C; MCF7: C; BxPC3: C; and PANC1: C. Cmpd 31: SKES: A; A4573:C; TC71: C; LNCap: C; PC3: C; MDA-MB-231: C; MCF7: C; BxPC3: C; andPANC1: C. Cmpd 32: SKES: A; A4573: C; TC71: C; LNCap: C; PC3: C;MDA-MB-231: C; MCF7: C; BxPC3: C; and PANC1: C. Cmpd 33: SKES: A; A4573:C; TC71: C; LNCap: C; PC3: C; MDA-MB-231: C; MCF7: C; BxPC3: C; andPANC1: C. Cmpd 34: SKES: A; A4573: C; TC71: C; LNCap: C; PC3: C;MDA-MB-231: C; MCF7: C; BxPC3: C; and PANC1: C. Cmpd 35: SKES: A; A4573:C; TC71: C; LNCap: C; PC3: C; MDA-MB-231: C; MCF7: C; BxPC3: C; andPANC1: C. Cmpd 36: SKES: C; A4573: C; TC71: C; LNCap: C; PC3: C;MDA-MB-231: C; MCF7: C; BxPC3: C; and PANC1: C. Cmpd 37: SKES: C; A4573:C; TC71: C; LNCap: C; PC3: C; MDA-MB-231: C; MCF7: C; BxPC3: C; andPANC1: C. Cmpd 38: SKES: C; A4573: C; TC71: C; LNCap: C; PC3: C;MDA-MB-231: C; MCF7: C; BxPC3: C; and PANC1: C. Cmpd 39: SKES: C; A4573:C; TC71: C; LNCap: C; PC3: C; MDA-MB-231: C; MCF7: C; BxPC3: C; andPANC1: C. A is IC₅₀>5 μM; B is IC₅₀<5 μM; and C is not determined. In axenograft study, A4573 tumor cells were implanted into mice. The micewere treated with certain compounds (oral bid). The mean volume of A4573tumors was measured at various times. Relative to vehicle control, cmp14 showed a 57% tumor growth inhibition (TGI) at 100 mg/kg bid, cmp 2did not show TGI at 200 mg/kg bid. In a similar rat xenograft study,cmpd 2 showed an 87% TGI, and cmpd 14 showed a 53% TGI, each compared tothe vehicle control.

Metabolic Activities of Certain Compounds

Metabolic activities of certain compounds were assayed with livermicrosomes using a NADPH regenerating system and standard protocols.Briefly, compounds were incubated with isolated human, rat, dog, ormouse liver microsomes. Reactions were initiated in 96-well plates byaddition of NADPH regenerating system (β-nicotinamide adeninedinucleotide phosphate; isocitric acid; and isocitric dehydrogenase).Reactions were quenched with cold acrylonitrile at 5, 10, 20, 30 and 60min, shaken and centrifuged at 4000 rpm for 20 min. Supernatantscontaining detected analyte compounds were analysed using LC/MS/MS withLC: Shimadzu LC 20-AD, MS: API4000, autosampler: CTC PAL; columns usedincluded CHIRALPAK AS-RH 150*4.6 mm, 5 μm Part No: ASRHCD-KK008, and Ace5 Phenyl, 50×2.1 mm, Part No. ACE-125-0502. Data analysis: T_(1/2) andCL were calculated use equations of first order kinetics: Ct=C₀*e^(−kt);C_(t)=(1/2)*C₀; t_(1/2)=In2/k=0.693/k; CL=V_(d)*k; and Vd=2 mL/mg.TABLES 5-8 summarise the results. In TABLES 5-8: R² is the correlationcoefficient of the linear regression for the determination of kineticconstant; T_(1/2) is half life; NCF is no cofactor.

TABLE 5 Human liver microsome Remaining Remaining T_(1/2) (T = 60 (NCF =60 Cmpd R² (min) min) min) 2 0.9771 20.2 12.7% 112.1% 3 0.7345 >14577.1% 112.6% 18 0.9869 23.0 17.8% 99.4% 13 0.9268 103.4 68.5% 105.1% 140.9901 27.7 22.8% 104.5% 37 0.9659 85.6 62.8% 103.4% 36 0.9972 23.817.9% 104.0% 39 0.9910 79.7 60.4% 100.0% 38 0.9970 18.7 11.2% 99.5%Testosterone 0.9928 18.9 10.9% 88.1% Diclofenac 0.9855 7.3 0.3% 77.8%Propafenone 0.9315 6.8 0.2% 92.5% Liver wt: 22 g/kg relative liverweight for human

TABLE 6 Rat liver microsome Remaining Remaining T_(1/2) (T = 60 (NCF =60 Cmpd R² (min) min) min) 2 0.9991 3.8 2.3% 92.7% 3 0.9989 7.1 2.7%92.6% 18 0.9995 1.3 0.0% 88.5% 13 0.9885 3.6 0.2% 97.0% 14 0.9971 2.80.1% 97.6% 37 0.9968 3.5 0.3% 95.4% 36 0.9943 2.6 0.2% 97.8% 39 0.98493.0 0.2% 90.5% 38 0.9137 2.9 0.2% 94.7% Testosterone 1.0000 1.0 0.0%87.3% Diclofenac 0.9985 13.3 4.1% 94.1% Propafenone 0.9926 2.0 0.0%89.2% Liver wt: 40 g/kg relative liver weight for rat

TABLE 7 Mouse liver microsome Remaining Remaining T_(1/2) (T = 60 (NCF =60 Cmpd R² (min) min) min) 2 0.9919 16.2 7.7% 90.2% 3 0.9902 7.4 0.4%97.5% 18 0.9820 9.6 1.6% 88.4% 13 0.9780 26.6 19.6% 85.6% 14 0.9474 23.119.7% 102.0% 37 0.9871 20.7 12.8% 88.6% 36 0.9904 11.9 3.0% 89.6% 390.9917 15.4 6.4% 89.7% 38 0.9968 6.8 0.0% 93.0% Testosterone 0.9992 3.30.0% 83.6% Diclofenac 0.9887 39.2 32.6% 88.1% Propafenone 0.9891 2.00.0% 91.7% Liver wt: 88 g/kg relative liver weight for mouse

TABLE 8 Dog liver microsome Remaining Remaining T_(1/2) (T = 60 (NCF =60 Cmpd R² (min) min) min) 2 0.9911 30.3 24.6% 100.6% 3 0.9977 21.314.0% 100.1% 18 0.9932 24.1 18.7% 92.9% 13 0.9935 8.4 0.7% 98.4% 140.9932 40.8 36.2% 97.1% 37 0.9855 9.1 1.0% 94.1% 36 0.9908 35.0 29.9%93.1% 39 0.9857 2.4 0.1% 96.3% 38 0.9961 33.5 28.3% 97.6% Testosterone0.9900 19.8 12.5% 91.0% Diclofenac 0.7395 >145 75.5% 89.6% Propafenone0.9765 5.2 0.1% 89.6% Liver wt: 32 g/kg relative liver weight for dog

Metabolic Stability of Certain Compounds

Half-life and clearance rates for certain compounds were assayed withhuman liver microsomes and human hepatocytes. Compounds were incubatedin the presence of human liver microsomes (or human hepatocytes) in a95% humidified incubator at 5% CO₂ to start the reactions. At each timepoint (0, 5, 15, 30, 60, 90 min) the reactions were stopped, vortexedand centrifuged. Supernatents were frozen until LC/MS/MS analysis. Theresults for human liver microsomes and human hepactocytes are summarizedin TABLE 9, and TABLE 10, respectively.

TABLE 9 Cmpd % remaining Half-life (min) CL_(hep) (mL/min/kg)* 1 5 10.317.28 12 18 18 15.69 7 49 >45 ~11.54 *hepatic clearance where blood flow= 20 mL/min/kg

TABLE 10 Cmpd % remaining Half-life (min) CL_(hep) (mL/min/kg)* 1 48 2268.7 12 56 >240 <8.7 7 100 >240 <8.7 *hepatic clearance where blood flow= 20 mL/min/kg

In another study, metabolism of compounds in hepatocytes from variousspecies was examined. Hepatocytes were contacted with a compound, andthe half-life of the compound was determined. The results are summarizedin TABLE 11.

TABLE 11 Half life of cmpds (min) Cmpd 1 18 14 7-Ethoxycoumarin7-Hydroxycoumarin Mouse 30.9 60.0 62.79 37.86 15.82 Rat 14.2 18.8 27.4138.24 12.41 Dog 16.7 30.6 20.26 9.14 9.38 Human 45.5 63.6 75.87 45.2920.78

Certain compounds were assayed with human liver microsomes (HLM) andhuman hepatocytes, and metabolites were detected. Briefly, metabolicstability was tested at one TA concentration (e.g., 1 μM) in duplicate.Loss of test article over time was evaluated in HLM (0.5 mg/mL) with andwithout NADPH at 0, 5, 10, 20, 30 and 45 min and hepatocytes (0.5×10⁶cells/mL) at 0, 15, 30, 60, 120 and 240 min. Positive control(diclofenac) and negative control (boiled HLM or heat inactivatedhepatocytes) were included. Diclofenac was monitored at incubation timessimilar to test article while the negative control was measured at 0 and45 min in HLM and 0 and 240 min in hepatocytes. Incubations were savedand used for metabolite identification using LC/MS/MS. The parentanalyte/internal standard peak area ratios was converted to percentagedrug remaining, using the time=0 peak area ratio values as 100%. Theslope of the linear regression from log percentage remaining versusincubation time relationships (−k) was determined by linear regressionfitting. From the individual log percentage remaining time profiles, themean half-life and intrinsic clearance were reported. UHPLC-HRMS orUHPLC-MS/MS experiment was performed on the authentic test articles tocheck for possible common fragment ions. Selected microsomal samplesfrom the 0, 10, 20, and 45 min aliquots, and the 0, 60, 120, and 240 minhepatocyte incubations aliquots were used for preliminary metaboliteidentification. The metabolites were summarized based on their massspectrometry peak areas. TABLE 12, TABLE 13 and TABLE 14 summariseresults for cmpds: 1, 12, and 7, respectively.

TABLE 12 [M + H] Retention Human liver Human Description (m/z) time(min) Structure microsomes hepatocytes Parent 366.0292 2.52

d d Demethylation + Glucuronidation 528.0482 1.97

nd d Oxidation 382.0249 2.24

d nd Demethylation 352.0141 2.27

d d d: detected, nd: not detected

TABLE 13 [M + H] Retention Human liver Human Description (m/z) time(min) Structure microsomes hepatocytes Parent 379.063 2.55

d d Demethylation + Oxidation 381.0406 2.13

d nd Di- demethylation 351.0305 2.25

d d Oxidation 395.0552 2.28

d nd d: detected, nd: not detected

TABLE 14 [M + H] Retention Human liver Human Description (m/z) time(min) Structure microsomes hepatocytes Parent 365.0458 2.40

d d Oxidation 381.0402 2.21

d nd Demethylation 351.0301 2.25

d d d: detected, nd: not detected

Pharmacokinetics of Certain Compounds

Certain pharmacokinetics parameters were determined for certaincompounds in vivo for intravenous and oral administration. Briefly,compounds were formulated and administered using IV bolus, continuous IVinfusion or oral administration. Blood was collected at multiple timepoints over 24 hours and processed into plasma. Plasma was analyses forparent using LC/MS/MS. Certain parameters for certain compounds wereobtained in duplicate. TABLE 15 summarises results with Cmp 14 for acontinuous infusion study in rat or dog. TABLE 16 summarises results forstudies in BALB/c mice or Sprague Dawley rats.

TABLE 15 Dose Vd CL MRT_(24-inf) CSS(_(3-24 h)) (mg/kg/day) T_(1/2) (h)k_(elim) (1/h) (mL/kg) (mL/h/kg) (h) (ng/mL) μM 24# 0.824 0.846 34572922 1.11 344 0.91 96# 1.00 0.703 4231 2976 1.73 1376 3.7 10{circumflexover ( )} 1.9 0.4 5643 2016 2.5 210 0.56 25{circumflex over ( )} 1.7 0.45663 2238 2.2 466 1.24 #: rat; {circumflex over ( )}: dog

TABLE 16 Dose (mg/kg), Cmax AUC T_(1/2) Tmax Cmpd route Vehicle (ng/mL)h*(ng/mL) %F (hr) (hr) 2 5, IV captisol 9381 1991 0.033 2 20, polabrasol/tetraglycol# 2243 3890 49 1 2 20, polabrasol/tetraglycol/water* 2835 4574 57 1 2 5, IV captisol 9381 1991 nd0.033 2 20, po labrasol/tetraglycol^(#) 2243 3896 40 1.6 1 2 20, polabrasol/tetraglycol/water* 2835 4574 47 1.5 1 11 5, IV captisol 123622102 0.033 11 20, po labrasol/tetraglycol# 1673 3783 45 1 11 20, polabrasol/tetraglycol/water* 1168 3828 46 0.5 11 5, IV captisol 123622871 0.8 0.033 11 20, po labrasol/tetraglycol^(#) 1673 3787 34 1 1 1120, po labrasol/tetraglycol/water* 1168 3832 34 0.8 0.5 5 5, IV captisol12993 2324 0.033 5 20, po labrasol/tetraglycol# 2179 5192 56 1 5 20, polabrasol/tetraglycol/water* 1569 4354 47 1 5 5, IV captisol 12993 32071.7 0.033 5 20, po labrasol/tetraglycol^(#) 2179 4939 47 3.1 0.5 5 20,po labrasol/tetraglycol/water* 1569 4081 39 2.8 0.5 14 5, IV captisol10891 4131 0.8 0.03 14 20, po labrasol/tetraglycol^(#) nd nd nd nd nd 1425, po labrasol/tetraglycol/water* 2772 13152 64 3.1 1 14 10, IVcaptisol 39800 6059 1.43 0.03 14 100, po labrasol/tetraglycol 32999146167 100 7.71 2 14 100, po PEG-400/tween 25967 92219 100 3.83 0 18 5,IV captisol 14343 3738 0.8 0.033 18 20, po labrasol/tetraglycol^(#) 32177497 50 1.4 1 18 20, po labrasol/tetraglycol/water* 6422 11029 74 0.9 1^(#)labrasol/tetraglycol: 9:1 *labrasol:tetraglycol:water is 72:8:20 nd:not determined Parameters determined in BALB/c mice or Sprague Dawleyrats

While the disclosure has been illustrated and described in detail in thedrawings and foregoing description, such illustration and descriptionare to be considered illustrative or exemplary and not restrictive. Thedisclosure is not limited to the disclosed embodiments. Variations tothe disclosed embodiments can be understood and effected by thoseskilled in the art in practicing the claimed disclosure, from a study ofthe drawings, the disclosure and the appended claims.

All references cited herein are incorporated herein by reference intheir entirety. To the extent publications and patents or patentapplications incorporated by reference contradict the disclosurecontained in the specification, the specification is intended tosupersede and/or take precedence over any such contradictory material.

Unless otherwise defined, all terms (including technical and scientificterms) are to be given their ordinary and customary meaning to a personof ordinary skill in the art, and are not to be limited to a special orcustomized meaning unless expressly so defined herein. It should benoted that the use of particular terminology when describing certainfeatures or aspects of the disclosure should not be taken to imply thatthe terminology is being re-defined herein to be restricted to includeany specific characteristics of the features or aspects of thedisclosure with which that terminology is associated.

Where a range of values is provided, it is understood that the upper andlower limit, and each intervening value between the upper and lowerlimit of the range is encompassed within the embodiments.

Terms and phrases used in this application, and variations thereof,especially in the appended claims, unless otherwise expressly stated,should be construed as open ended as opposed to limiting. As examples ofthe foregoing, the term ‘including’ should be read to mean ‘including,without limitation,’ ‘including but not limited to,’ or the like; theterm ‘comprising’ as used herein is synonymous with ‘including,’‘containing,’ or ‘characterized by,’ and is inclusive or open-ended anddoes not exclude additional, unrecited elements or method steps; theterm ‘having’ should be interpreted as ‘having at least;’ the term‘includes’ should be interpreted as ‘includes but is not limited to;’the term ‘example’ is used to provide exemplary instances of the item indiscussion, not an exhaustive or limiting list thereof; adjectives suchas ‘known’, ‘normal’, ‘standard’, and terms of similar meaning shouldnot be construed as limiting the item described to a given time periodor to an item available as of a given time, but instead should be readto encompass known, normal, or standard technologies that may beavailable or known now or at any time in the future; and use of termslike ‘preferably,’ ‘preferred,’ ‘desired,’ or ‘desirable,’ and words ofsimilar meaning should not be understood as implying that certainfeatures are critical, essential, or even important to the structure orfunction of the invention, but instead as merely intended to highlightalternative or additional features that may or may not be utilized in aparticular embodiment of the invention. Likewise, a group of itemslinked with the conjunction ‘and’ should not be read as requiring thateach and every one of those items be present in the grouping, but rathershould be read as ‘and/or’ unless expressly stated otherwise. Similarly,a group of items linked with the conjunction ‘or’ should not be read asrequiring mutual exclusivity among that group, but rather should be readas ‘and/or’ unless expressly stated otherwise.

With respect to the use of substantially any plural and/or singularterms herein, those having skill in the art can translate from theplural to the singular and/or from the singular to the plural as isappropriate to the context and/or application. The varioussingular/plural permutations may be expressly set forth herein for sakeof clarity. The indefinite article “a” or “an” does not exclude aplurality. The mere fact that certain measures are recited in mutuallydifferent dependent claims does not indicate that a combination of thesemeasures cannot be used to advantage. Any reference signs in the claimsshould not be construed as limiting the scope.

It will be further understood by those within the art that if a specificnumber of an introduced claim recitation is intended, such an intentwill be explicitly recited in the claim, and in the absence of suchrecitation no such intent is present. For example, as an aid tounderstanding, the following appended claims may contain usage of theintroductory phrases “at least one” and “one or more” to introduce claimrecitations. However, the use of such phrases should not be construed toimply that the introduction of a claim recitation by the indefinitearticles “a” or “an” limits any particular claim containing suchintroduced claim recitation to embodiments containing only one suchrecitation, even when the same claim includes the introductory phrases“one or more” or “at least one” and indefinite articles such as “a” or“an” (e.g., “a” and/or “an” should typically be interpreted to mean “atleast one” or “one or more”); the same holds true for the use ofdefinite articles used to introduce claim recitations. In addition, evenif a specific number of an introduced claim recitation is explicitlyrecited, those skilled in the art will recognize that such recitationshould typically be interpreted to mean at least the recited number(e.g., the bare recitation of “two recitations,” without othermodifiers, typically means at least two recitations, or two or morerecitations). Furthermore, in those instances where a conventionanalogous to “at least one of A, B, and C, etc.” is used, in generalsuch a construction is intended in the sense one having skill in the artwould understand the convention (e.g., “a system having at least one ofA, B, and C” would include but not be limited to systems that have Aalone, B alone, C alone, A and B together, A and C together, B and Ctogether, and/or A, B, and C together, etc.). In those instances where aconvention analogous to “at least one of A, B, or C, etc.” is used, ingeneral such a construction is intended in the sense one having skill inthe art would understand the convention (e.g., “a system having at leastone of A, B, or C” would include but not be limited to systems that haveA alone, B alone, C alone, A and B together, A and C together, B and Ctogether, and/or A, B, and C together, etc.). It will be furtherunderstood by those within the art that virtually any disjunctive wordand/or phrase presenting two or more alternative terms, whether in thedescription, claims, or drawings, should be understood to contemplatethe possibilities of including one of the terms, either of the terms, orboth terms. For example, the phrase “A or B” will be understood toinclude the possibilities of “A” or “B” or “A and B.”

All numbers expressing quantities of ingredients, reaction conditions,and so forth used in the specification are to be understood as beingmodified in all instances by the term ‘about.’ Accordingly, unlessindicated to the contrary, the numerical parameters set forth herein areapproximations that may vary depending upon the desired propertiessought to be obtained. At the very least, and not as an attempt to limitthe application of the doctrine of equivalents to the scope of anyclaims in any application claiming priority to the present application,each numerical parameter should be construed in light of the number ofsignificant digits and ordinary rounding approaches.

Furthermore, although the foregoing has been described in some detail byway of illustrations and examples for purposes of clarity andunderstanding, it is apparent to those skilled in the art that certainchanges and modifications may be practiced. Therefore, the descriptionand examples should not be construed as limiting the scope of theinvention to the specific embodiments and examples described herein, butrather to also cover all modification and alternatives coming with thetrue scope and spirit of the invention.

What is claimed is:
 1. A compound having a structure of Formula (I):

or a stereoisomer, a pharmaceutically acceptable salt, or solvatethereof, wherein R₁, R₂, R₃, and R₄ are independently selected from thegroup consisting of H, Cl, —CN and —CF₃; wherein A is selected from thegroup consisting of H and C₁₋₆ alkyl; wherein D is selected from thegroup consisting of —OH and —O(C₁₋₆ alkyl); wherein R₅ and R₆ areindependently selected from the group consisting of H, F, and C₁₋₆alkyl, or wherein R₅ and R₆ taken together form a substituted orunsubstituted cycloalkyl ring; wherein R₁₂ is independently selectedfrom the group consisting of C₃₋₈ cycloalkyl and

wherein R₇, R₈, R₉, R₁₀ and R₁₁ are independently selected from thegroup consisting of H, halogen, CN, CF₃, C₁₋₆ alkyl, aryl, heteroaryl,—O(aryl), —O(heteroaryl), —CO₂H, —CO₂(C₁₋₆ alkyl), —NHSO₂(C₁₋₆ alkyl),—NHSO₂(aryl), —NHCONH(C₁₋₆ alkyl), —NHCON(C₁₋₆ alkyl)₂, —N(C₁₋₆alkyl)CONH₂, alkyl)CONH(C₁₋₆ alkyl), —N(C₁₋₆ alkyl)CON(C₁₋₆ alkyl)₂,—SO₂(C₁₋₆ alkyl), —SO₂NH₂, —SO₂NH(C₁₋₆ alkyl), —SO₂N(C₁₋₆ alkyl)₂, C₃₋₈cycloalkyl, and C₃₋₈ heterocycloalkyl.
 2. The compound of claim 1,wherein R₉ is selected from the group consisting of aziridinyl,azetidinyl, pyrrolidinyl, and morpholinolyl.
 3. The compound of claim 1,wherein R₉ is selected from the group consisting of isopropyl andcyclopropyl.
 4. The compound of claim 1, having a structure of Formula(Ia):

or a stereoisomer, a pharmaceutically acceptable salt, or solvatethereof, wherein R₁, R₂, R₃, and R₄ are independently selected from thegroup consisting of H and Cl; wherein R₇, R₈, R₁₀ and R₁₁ areindependently selected from the group consisting of H and halogen; andwherein R₉ is independently selected from the group consisting C₃₋₈cycloalkyl and C₃₋₈ heterocycloalkyl.
 5. The compound of claim 1,wherein R₁ and R₄ are Cl and R₂ and R₃ are H.
 6. The compound of claim1, selected from the group consisting of:

or a stereoisomer, a pharmaceutically acceptable salt, ester, or solvatethereof.
 7. The compound of claim 6, selected from the group consistingof:

or a stereoisomer, a pharmaceutically acceptable salt, ester, or solvatethereof.
 8. The compound of claim 1, selected from the group consistingof:

or a stereoisomer, a pharmaceutically acceptable salt, ester, or solvatethereof.
 9. The compound of claim 1, selected from the group consistingof:

or a stereoisomer, a pharmaceutically acceptable salt, ester, or solvatethereof.
 10. The compound of claim 1, having the structure:

or a stereoisomer, a pharmaceutically acceptable salt, ester, or solvatethereof.
 11. The compound of claim 1, having the structure:

or a stereoisomer, a pharmaceutically acceptable salt, ester, or solvatethereof.
 12. The compound of claim 1, having the structure:

or a stereoisomer, a pharmaceutically acceptable salt, ester, or solvatethereof.
 13. A pharmaceutical composition comprising the compound ofclaim 1 and a pharmaceutically acceptable carrier.
 14. A method forinhibiting proliferation of a cell, wherein the cell overexpresses anETS gene or comprises an ETS fusion gene, comprising contacting the cellwith an effective amount of the compound of claim
 1. 15. The method ofclaim 14, wherein the ETS gene or the ETS fusion gene is selected fromthe group consisting of FLI1, ERG, ETV1, and ETV4.
 16. The method ofclaim 14, wherein the cell is a cancer cell, wherein the cancer isselected from the group consisting of Ewing's sarcoma, prostate cancer,glioblastoma, acute myeloid leukemia, breast cancer, head cancer, neckcancer, melanoma, non-small cell lung cancer, ovarian cancer, anduterine cancer.
 17. The method of claim 14, wherein the cell ismammalian.
 18. A method of killing or inhibiting the growth of aneoplastic cell, comprising contacting the cell with an effective amountof the compound of claim
 1. 19. The method of claim 18, wherein the cellis a cancer cell, wherein the cancer is selected from the groupconsisting of Ewing's sarcoma, prostate cancer, glioblastoma, acutemyeloid leukemia, breast cancer, head cancer, neck cancer, melanoma,non-small cell lung cancer, ovarian cancer, and uterine cancer.
 20. Themethod of claim 18, wherein the cell is mammalian.